Methods for diagnosis and intervention of hepatic disorders

ABSTRACT

The disclosure provides a method for quantification of hepatic function in a subject comprising measuring the clearance of an orally administered isotopically labeled cholic acid in a subject with, or suspected of having or developing, a hepatic disorder, for example, chronic hepatitis C. The disclosure further provides methods and kits for assessment of hepatic function.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/195,762, filed Jun. 28, 2016, now U.S. Pat. No. 10,215,746, which isa continuation of U.S. application Ser. No. 14/273,085, filed May 8,2014, issued as U.S. Pat. No. 9,417,230 on Aug. 16, 2016, which is adivisional of U.S. application Ser. No. 12/557,916, filed Sep. 11, 2009,issued as U.S. Pat. No. 8,778,299 on Jul. 15, 2014, which is acontinuation-in-part of U.S. application Ser. No. 11/814,793, issued asU.S. Pat. No. 8,613,904 on Dec. 24, 2013, with a § 371 date of May 13,2008, which is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/US06/03132, which has an Internationalfiling date of Jan. 26, 2006, which designated the United States ofAmerica and which claims the benefit of U.S. Provisional ApplicationSer. No. 60/647,689, filed Jan. 26, 2005, the entire contents of each ofwhich is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Contract No.DK092327 and Grant No. RR000051 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Chronic hepatitis C affects 4 million patients in the United States, andresults in 10,000 deaths annually. Major clinical consequences ofchronic liver disease are related to the effect of hepatic fibrosis inproducing portal hypertension and in the progressive decline of thefunctioning hepatic mass. Currently, measuring clearance rates ofsubstances primarily removed from circulation by the liver provides themost sensitive, non-intrusive and specific indicator of liver function.

In humans, the two primary bile acids synthesized by the liver arecholic acid and chenodeoxycholic acid, which are converted intosecondary bile acids by intestinal bacteria. These bile acids areconjugated with glycine or taurine and secreted by the liver. Serum bileacid levels are determined by the balance between intestinal absorptionand hepatic elimination of bile acid.

Cholic acid is an example of a model bile acid. Orally administeredcholic acid is absorbed across the epithelial lining cells of the smallintestine, bound to albumin in the portal blood, and transported to theliver via the portal vein. Approximately 80 to 85% of cholic acid isextracted from the portal blood in its first pass through the liver.Cholic acid that escapes hepatic extraction exits the liver via hepaticveins that drain into the vena cava back to the heart, and is deliveredto the systemic circulation. The area under the curve (AUC) ofperipheral venous concentration versus time after oral administration ofcholic acid quantifies the fraction of cholic acid escaping hepaticextraction and defines “oral cholate clearance”.

Intravenously administered cholic acid, bound to albumin, distributessystemically and is delivered to the liver via both portal venous andhepatic arterial blood flow. The AUC of peripheral venous concentrationversus time after intravenous administration of cholic acid isequivalent to 100% systemic delivery of cholic acid. The ratio of theAUCs of orally to intravenously administered cholic acid, corrected foradministered doses, defines cholate shunt.

After uptake by the liver, cholic acid is efficiently conjugated toeither glycine or taurine and secreted into bile. Physicochemicallycholic acid is easily separated from other bile acids and bile acid orcholic acid conjugates, using chromatographic methods.

An NIH-sponsored Hepatitis C Antiviral Long-Term Treatment againstCirrhosis (HALT-C) Trial examined whether long-term use of antiviraltherapy (maintenance treatment) would slow the progression of liverdisease. In noncirrhotic patients who exhibited significant fibrosis,effective maintenance therapy was expected to slow or stop histologicalprogression to cirrhosis as assessed by serial liver biopsies. However,tracking disease progression with biopsy carries risk of complication,possibly death. In addition, sampling error and variation of pathologicinterpretation of liver biopsy limits the accuracy of histologicassessment and endpoints. The histologic endpoint is less reliablebecause advanced fibrosis already exists and changes in fibrosis relatedto treatment or disease progression cannot be detected. Thus, standardendpoints for effective response to maintenance therapy in cirrhoticpatients are prevention of clinical decompensation (ascites, varicealhemorrhage, and encephalopathy) and stabilization of liver function asmeasured clinically by Childs-Turcotte-Pugh (CTP) score. However,clinical endpoints and CTP score are insensitive parameters of diseaseprogression.

In one proposal, studies were designed to analyze disease progression ina unique subset of patients with chronic hepatitis C, those withfibrosis and early, compensated cirrhosis. These patients arecharacterized by absence of clinical findings and normal or nearlynormal values for standard routine biochemical parameters includingserum albumin and prothrombin time. Child-Turcotte-Pugh scores willrange from 5 to 6. For this reason, this subgroup of patients maybenefit from quantitative tests of liver function that might be moreuseful than standard biochemical measurements, and more sensitive thanclinical endpoints for evaluating the degree and progression of hepaticdysfunction.

Because early intervention of liver dysfunction is critical, a needexists for the detection of early signs that predict the onset orprogression of a condition. A number of critical needs could be met byeffective and accurate tests of hepatic function.

SUMMARY OF THE INVENTION

The disclosure provides a method for assessment and quantification ofhepatic function in a subject comprising measuring the clearance of anorally administered isotopically labeled cholic acid to a subjectsuspected of having or developing a hepatic disorder, for example,chronic hepatitis C. The disclosure further provides methods and kitsfor assessment of hepatic function.

In one embodiment, the disclosure provides a method for assessment ofhepatic function in a subject, the method comprising: administeringorally an isotopically labeled cholic acid to a subject with, orsuspected of having or developing, a hepatic disorder, wherein noadditional cholic acid compound is intravenously co-administered;collecting samples from the subject over intervals for a period of lessthan 3 hours after administration of the agents to the subject; andmeasuring the clearance of the orally administered isotopically labeledcholic acid as an indicator of hepatic function in the subject. In oneaspect, the isotopically labeled cholate is selected from 24-¹³C cholicacid and 2,2,4,4-²H cholic acid. In another aspect, the samplescollected over an interval period of time comprise blood or serumsamples. In another aspect, the samples are collected over a period ofabout 90 minutes or less. In a specific aspect, blood samples collectedfrom the subject at 5, 20, 45, 60 and 90 minutes post-administration. Inone aspect, the measuring step comprises quantifying the isotopicallylabeled cholic acid in the samples by GC-MS or HPLC-MS.

In another embodiment, the disclosure provides a method for assessmentof hepatic function in a subject that comprises oral cholate clearanceand at least one additional hepatic assessment test. In certain aspects,the additional hepatic assessment test is selected from clearance ormetabolism of aminopyrine, clearance or metabolism of aminopyrine,clearance or metabolism of antipyrine, clearance or metabolism of bileacids other than cholate, clearance or metabolism of caffeine, clearanceof or metabolism erythromycin, clearance or metabolism of nitroglycerin,clearance of or metabolism galactose, clearance or metabolism ofindocyanine green, clearance or metabolism of lidocaine, clearance ormetabolism of midazolam, clearance or metabolism of omeprazole,clearance or metabolism of dextromethorphan, clearance or metabolism ofphenacetin, clearance or metabolism of methacetin, liver-spleen scan,serum bilirubin analysis, alanine aminotransferase analysis, aspartateaminotransferase analysis, and alkaline phosphatase analysis. In afurther aspect, the additional hepatic assessment test is amulti-isotope test. In another aspect, the additional hepatic assessmenttest is a quantitative liver function test (QLFT). The quantitativeliver function test (QLFT) is selected from a hepatic metabolic functiontest, a hepatic blood flow test or a combination of these tests. In aspecific aspect, the hepatic metabolic function test is a multi-isotopecaffeine test which comprises administering distinguishable caffeinesolutions orally, intravenously or in combination. In a specific aspect,the administering step comprises administering three distinguishablecaffeine solutions to a subject at different time intervals. In onespecific aspect, a single sample is obtained sometime afteradministration of the distinguishable caffeine solutions to assess thehepatic condition of the subject.

In one embodiment, the clearance of the isotopically labeled cholic acidis used as an indication of a need for at least one therapeutictreatment of the subject with a hepatic disorder. In one aspect, thetherapeutic treatment comprises an antiviral therapy. In another aspect,the hepatic disorder comprises chronic hepatitis C. In anotherembodiment, the clearance of the isotopically labeled cholic acid isused for assessment of the progression of at least one hepatic conditionin a subject.

In one embodiment, the disclosure provides a method for determination ofcholate shunt. This method can be used for assessment of the progressionof at least one hepatic condition in a subject, the method comprising:administering orally a first distinguishable isotopically labeled cholicacid to a subject having, or suspected of having or developing, ahepatic disorder; co-administering intravenously a seconddistinguishable isotopically labeled cholic acid to the subject;collecting blood or serum samples over intervals for a period of lessthan 3 hours after administration of the agents to the subject;quantifying the first and the second isotopically labeled cholic acidsin the samples by HPLC-MS; and calculating the cholate shunt using theformula: AUCoral/AUCiv×Doseiv/Doseoral×100%; wherein AUCoral is the areaunder the curve of the serum concentrations of the first cholic acid andAUCiv is the area under the curve of the second cholic acid; and whereinthe cholate shunt is an indicator of the progression of at least onehepatic disorder of the subject. In one aspect, the orally administeringof the first labeled cholic acid and the intravenously co-administeringof the second labeled cholic acid are performed simultaneously. Inanother aspect, the collecting step comprises collecting samples over aperiod of about 90 minutes or less. In a further aspect, the samplescomprise blood or serum samples collected from the subject at 5, 20, 45,60 and 90 minutes post-dose.

In one aspect, the cholate shunt is an indicator of a need for at leastone therapeutic treatment of the subject with a hepatic disorder. In afurther aspect, the at least one therapeutic treatment comprises anantiviral therapy. In another aspect, the hepatic disorder compriseschronic hepatitis C.

In another embodiment, the disclosure provides a method for assessmentof hepatic function in a subject that comprises cholate shunt and atleast one additional hepatic assessment test. In certain aspects, the atleast one additional hepatic assessment test is selected from clearanceor metabolism of aminopyrine, clearance or metabolism of aminopyrine,clearance or metabolism of antipyrine, clearance or metabolism of bileacids other than cholate, clearance or metabolism of caffeine, clearanceof or metabolism erythromycin, clearance or metabolism of nitroglycerin,clearance of or metabolism galactose, clearance or metabolism ofindocyanine green, clearance or metabolism of lidocaine, clearance ormetabolism of midazolam, clearance or metabolism of omeprazole,clearance or metabolism of dextromethorphan, clearance or metabolism ofphenacetin, clearance or metabolism of methacetin, liver-spleen scan,serum bilirubin analysis, alanine aminotransferase analysis, aspartateaminotransferase analysis, and alkaline phosphatase analysis. In anotheraspect, the additional hepatic assessment test is a quantitative liverfunction test (QLFT), which may be a hepatic metabolic function test, ahepatic blood flow test or combination thereof.

In another embodiment, the disclosure provides a kit of components fordetermining one or both of cholate clearance and cholate shunt in asubject with, or suspected of having or developing, a hepatic disorder;the kit comprising a first component comprising one or more vials, eachvial comprising a single oral dose of a first distinguishable cholatecompound; and a second component comprising one or more vials, each vialcomprising a single intravenous dose of a second distinguishable cholatecompound. In one aspect, the kit further comprises a third componentcomprising one or more vials, each vial comprising a quantity of humanserum albumin for mixing with a single intravenous dose of the seconddistinguishable cholate compound prior to intravenous administration. Inanother aspect, the kit further comprises one or more sets of labeledsterile blood-serum sample collection tubes. In a further aspect, thekit also comprises one or more sets of labeled transport vials, eachtransport vial containing an internal cholic acid standard. In onespecific aspect, the kit also comprises a single box for both shippingthe vials to a health care practitioner and shipping the samples fromthe health care practitioner to a reference lab for analysis. In anotherspecific aspect, the first distinguishable cholate compound is2,2,4,4-²H cholic acid and the second distinguishable agent is 24-¹³Ccholic acid. In a further specific aspect, the ²H-cholic acid is in apowder form or in a solution form.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1 illustrates an exemplary computing device.

FIG. 2 illustrates an cholate oral clearance curve derived from anaverage sampling of over 300 patients administered cholate orally.

FIG. 3 illustrates a cholate IV clearance curve derived from an averageof samples of over 300 patients administered cholate intravenously.

FIG. 4 illustrates a flow chart of an exemplary model curve derivationalgorithm 400 having exemplary operations for generating a modelclearance curve in accordance with one embodiment of the presentinvention.

FIG. 5 illustrates a flow chart of an exemplary model IV clearance curvederivation algorithm 500 for generating a model IV clearance curve basedon a standard 14 point IV clearance curve.

FIG. 6 illustrates a flow chart of an exemplary embodiment of analgorithm for deriving a model oral clearance curve based on a standardfourteen point oral clearance curve.

FIG. 7 illustrates an average oral clearance curve derived from asampling of over 300 patients showing a model five point oral clearancecurve and a standard fourteen point clearance curve from which the modelcurve was derived.

FIG. 8 shows mean differences (+/−s.d.) of measurements of cholate shuntusing reduced numbers of time points in comparison to the standard 14time point method.

FIG. 9 shows orally administered cholate clearance and cholate shunt atbaseline and post-SVR. Sustained virological response was associatedwith a 32% increase in cholate oral clearance, a measure or portal bloodflow, as shown in FIG. 9(a). Sustained virological response was alsoassociated with a 26% decrease in cholate shunt, a measure ofportal-systemic shunting, as shown in FIG. 9(b).

FIG. 10 shows the percentage change between baseline and follow-upstudies for various QLFTs. The black bars depict the changes after SVR,and the grey bars show the changes in patients with nonresponse (NR).

FIG. 11 shows average cholate shunt values and the number of hepatitis Cpatients at each time interval who experienced first clinical outcome,or no outcome, in the periods 0 to 24 months, 24 to 48 months and after48 months.

FIG. 12 shows the cutoff for cholate shunt that predicts the 2-year riskof decompensation in hepatitis C patients.

FIG. 13 shows average oral cholate clearance values and the number ofhepatitis C patients at each time interval who experienced firstclinical outcome, or no outcome, in the periods 0 to 24 months, 24 to 48months and after 48 months.

FIG. 14 shows the cutoff for oral cholate clearance that predicts the2-year risk of decompensation.

FIG. 15 shows the correlation between mean cholate shunt and Ishak stageof fibrosis for Hepatitis-C patients (n=282).

DETAILED DESCRIPTION OF THE INVENTION

In the following section, several methods are described to detailvarious embodiments of the invention. It will be obvious to one skilledin the art that practicing the various embodiments does not require theemployment of all or even some of the specific details outlined herein,but rather that concentrations, times and other specific details may bemodified through routine experimentation. In some cases, well knownmethods or components have not been included in the description in orderto prevent unnecessary masking of various embodiments.

Definitions and Acronyms

As used herein, “a” or “an” may mean one or more than one of an item. Asused herein “clearance” may mean the removing of a substance from oneplace to another. As used herein the specification, “subject” or“subjects” may include but are not limited to mammals such as humans ormammals for example dogs, cats, ferrets, rabbits, pigs, horses, cattleto birds, or reptiles. Other definitions are provided throughout thespecification.

The acronym “HALT-C” refers to the Hepatitis C Antiviral Long-termTreatment against Cirrhosis trial. The HALT-C trial was a large,prospective, randomized, controlled trial of long-term low dose peginterferon therapy in patients with advanced hepatitis C who had not hada sustained virologic response to a previous course of interferon-basedtherapy.

The acronym “IV” or “iv” refers to intravenous.

The acronym “PO” refers to per oral.

The acronym “PHM” refers to perfused hepatic mass.

The acronym “MEGX” refers to monoethylglycinexylidide, a metabolite oflidocaine.

The acronym “SF” refers to shunt fraction, for example, as in cholateSF.

The acronym “Cl_(oral)” refers to clearance of an orally administeredcholate compound.

The acronym “Cl_(iv)” refers to clearance of an intravenouslyadministered cholate compound.

The acronym “ROC” refers to receiver operator curve.

The acronym “QLFT” refers to Quantitative Liver Function Test.

The term “Ishak Fibrosis Score” is used in reference to a scoring systemthat measures the degree of fibrosis (scarring) of the liver, which iscaused by chronic necroinflammation. A score of 0 represents nofibrosis, and 6 is established fibrosis. Scores of 1 and 2 indicatedegrees of portal fibrosis; stages 3 and 4 indicate bridging fibrosis. Ascore of 5 indicates nodular formation and incomplete fibrosis.

In one embodiment, Quantitative Liver Function Tests (QLFTs), such asassays that measure the liver's ability to metabolize or extract testcompounds, can identify patients with impaired hepatic function atearlier stages of disease, and possibly define risk for cirrhosis,splenomegaly, and varices. One of these assays is the cholate shuntassay where the clearance of cholate is assessed by analyzing bodilyfluid samples after exogenous cholate has been taken up by the body.

In one aspect, QLFTs can be used to measure hepatic disease progression.Investigators have used the clearance or measurement of metabolites ofaminopyrine, antipyrine, bile acids, propranolol, midazolam,dextromethorphan, methionine, methoximine, caffeine, erythromycin,galactose, indocyanine green, lidocaine, phenacetin and methacetin toassess hepatic function. In certain aspects, multiple isotopes of any ofthese compounds may be utilized to assess hepatic function. For example,differentiable isotopes of a particular compound can be administered bydifferent routes of administration. Clearances of test compounds aretypically defined as dependent upon either hepatic metabolism(aminopyrine, antipyrine, caffeine, erythromycin) or hepatic blood flow(bile acids, indocyanine green).

Each quantitative test has advantages and disadvantages over other testsand few studies have compared multiple tests within the same cohort ofpatients. Studies herein such as QLFTs in HALT-C patients represent acomprehensive comparison of 12 QLFTs, using 8 test compounds, in thesame patient. Also, these studies represent patients with chronichepatitis C and advanced fibrosis using quantitative tests to predictoutcome and measuring changes in hepatic function over prolonged periodsof time (e.g. 4-6 years).

The most commonly used quantitative tests assess hepatic metaboliccapacity. Aminopyrine (dimethylaminoantipyrine) is metabolized primarilyby N-demethylation. The hepatic capacity to metabolize aminopyrine canbe measured from the specific activity of [¹⁴CO₂] in breath samplesobtained two hours after oral administration of a tracer dose of [¹⁴C]aminopyrine. A related compound, antipyrine, is extensively metabolizedby a group of cytochrome P450 dependent liver microsomal enzymes; only5% of the drug appears unchanged in the urine. The plasma or salivarydisappearance of antipyrine follows first order kinetics and obeys asimple, one-compartment model. As with all drugs whose clearance isprimarily dependent upon metabolism, elimination is not greatlyinfluenced by changes in hepatic blood flow. The main problem with useof these compounds is the reported low rate of severe bone marrowdepression, including anemia. There has been one reported fatality dueto an overwhelming hypersensitivity reaction in response to a singledose. Antipyrine is also not readily available for use in humans.

Phenacetin differs from aminopyrine in that its metabolism is mediatedby cytochrome P448 and the [¹⁴C] phenacetin breath test is anothertechnique to measure hepatic function in humans.

Methacetin can also be used to evaluate hepatic metabolism. For example,a [15N]methacetin urine test can be used to study human 0-demethylaseactivity to characterize hepatic detoxification capacity following oraladministration of an aqueous solution. (Krumbiegel at al.,[¹⁵N]Methacetin urine test: A method to study the development of hepaticdetoxification capacity, 1990, European J. Pediatrics, 149(6); 393-395).In another example, a continuous [¹³C] methacetin breath test (13C-MBT,BreathID®) can be used for assessment of hepatic microsomal enzymefunction (cytochrome P450 CYP1A2) following oral administration ofmethacetin. (Lalazar et al., A Continuous 13C Methacetin Breath Test forNoninvasive Assessment of Intrahepatic Inflammation and Fibrosis inPatients with Chronic HCV Infection and Normal ALT., 2008, J. ViralHepatitis, 15(10):716-728).

Caffeine has been used as a test compound for quantitative assessment ofthe liver because of its relative lack of toxicity, rapid absorption,its complete metabolism by the liver, and its ready availability.Caffeine is eliminated by first order kinetics but pathways ofmetabolism are sometimes extensive and complex. A disadvantage ofprevious caffeine tests is that caffeine is ubiquitously found in a widevariety of commonly ingested foodstuffs, supplements, and medications;ingestion of caffeine from these sources typically invalidates resultsof most standard caffeine assays. In addition, the metabolism andclearance of caffeine can be altered by coadministration of drugs ormedications.

Erythromycin is eliminated primarily by n-demethylation by hepaticcytochrome P450 enzymes, predominantly CYP3A4 (cytochrome P450 3A4).Numerous xenobiotics, including up to 50% of prescribed medications, aremetabolized through the CYP3A4 pathway and may enhance or inhibiterythromycin clearance and metabolism. These effects invalidate the useof erythromycin as a liver function test.

Galactose elimination is complicated by extrahepatic metabolism.Approximately 60% of the total plasma elimination of galactose after asingle intravenous injection is due to hepatic clearance; the remaining40% is due to distribution and metabolism of galactose outside theliver. Thus, galactose elimination capacity is only partially a liverfunction test.

Other Tests Assess Flow-Dependent Hepatic Clearance.

Indocyanine green (ICG), when administered intravenously is removed fromthe circulation by the liver with a first-pass hepatic extraction up to80%. After uptake by the liver, indocyanine green is transported to bilewithout metabolic transformation. However, as is true of otherintravenously administered test compounds, ICG is insensitive and cannotdetect early stage disease or small changes in the hepatic condition.

Lidocaine is initially cleared by the liver in a flow-dependent fashion;first pass elimination is up to 81%. Once taken up by the liver,lidocaine is metabolized by oxidative N-demethylation (cytochrome P4503A4) to monoethylglycinexylidide (MEGX). MEGX concentrations are aresult of rapid hepatic uptake and clearance from the blood followed byhepatic metabolism and have been used to assess hepatic function inpotential liver donors, in liver transplant recipients, and inpredicting survival in patients with cirrhosis. Early results suggestthat lidocaine-MEGX is useful in assessing short-term prognosis incirrhotic patients independent of the cause of the underlying liverdisease. However, MEGX is subject to the same concerns raised above forintravenously administered compounds (ICG) and its blood level may beaffected by interference from coadministered medications, supplements,or dietary factors.

Bile acids are naturally-occurring compounds that exhibit flow-dependenthepatic clearance. Dual isotope techniques allow measurement offirst-pass hepatic elimination of bile acids from the portalcirculation. Flow-dependent, first pass elimination of bile acids by theliver ranges from 60% for unconjugated dihydroxy, bile acids to 95% forglycine-conjugated cholate. Free cholate, used herein has a reportedfirst-pass elimination of approximately 80% which agrees closely withobserved first pass elimination in healthy controls of about 83%. Plasmaclearance of oral and intravenous cholic acid in subjects with andwithout chronic liver disease were studied. These studies demonstratedreduced clearance of cholate in patients who had either hepatocellulardamage or portosystemic shunting.

Liver-spleen scans are an effective measure of many parameters affectedby chronic liver disease. The liver-spleen scan is useful when theparameters measured are given quantitative expression by SPECT analysis.These parameters can include: 1) precise measurement of sulfur colloiddistribution, 2) organ volumes functional 3) organ volumes and/ornon-functional volume ratios. Sulfur colloid distribution is determinedby Kupffer cell extraction of sulfur colloid and hepatic blood flow.Increased sulfur colloid distribution to spleen and bone marrow is dueto either decreased hepatic extraction or decreased hepatic perfusion,both of which are determined by hepatic fibrosis. Thus, precisemeasurement of this distribution from planar measurement as aredistribution ratio (RR) or from volumetric parameters such as theperfused hepatic mass (PHM) correlates with ICG clearance and othertests of hepatocyte function. In one embodiment, any liver-spleen scantechnique known in the art may be combined with any metabolic orclearance assay disclosed herein.

Typically, the PHM remains normal (>100) as scar tissue builds up in theliver until cirrhosis is well established. Once cirrhosis is establishedthe PHM measurement deteriorates proportional to liver disease severity.For example, the PHM range is below the normal range (PHM=100-120) withcompensated cirrhosis (PHM=80-110), lower still with ascites andvariceal bleeding (PHM=40-80), and generally less than sixty incirrhotic patients who die or require transplant. The non-fibrotic mass(functional hepatic mass) in a group of cirrhotic patients whose liverwas removed at transplant or autopsy correlated closely with the PHM(correlation coefficient 0.95).

Areas of critical need for noninvasive QLFTs include, but are notlimited to the following: detection of fibrosis and early cirrhosis(e.g. to avoid liver biopsy); detection of risk of varices (e.g.identification of patients who might benefit from endoscopy therapy);assessment of likelihood to respond to antiviral therapy (e.g. morerefined selection of patients for treatment); defining level of hepaticimpairment prior to treatments that might affect or could be affected byliver function (e.g. more precise definition of the level of hepaticimpairment); selection of patients for transjugular intrahepaticportal-systemic shunt (TIPS) placement, or defining impairment prior toinstitution of chemotherapeutic agents to treat cancer; tracking diseaseprogression (e.g., early detection of decompensation); and measurementof the effects of therapies or interventions (e.g., the changes in QLFTsmay occur long before clinical deterioration and QLFTs would haveincreased sensitivity at detecting changes in the hepatic conditioninduced by the treatment/intervention; therefore, a smaller sample sizecould be utilized in defining effects).

Non-invasive tests have been used for prediction of fibrosis orcirrhosis. Methods include models using standard laboratory tests andother blood components, clearance of test compounds, Dopplerultrasonography, transient ultrasonographic elastography and magneticresonance elastography. However, none of these methods accuratelyquantifies the portal circulation or portal-systemic shunting. Incontrast, the cholate method disclosed herein not only quantifies theportal circulation and portal-systemic shunting, but also correlateswell with cirrhosis, varices and variceal size.

In HALT-C patients, cholate Cl_(oral) and cholate shunt comparedfavorably to other quantitative tests of liver function. They correlatedbetter with cirrhosis, varices, standard laboratory tests and responseto antiviral therapy than caffeine elimination, antipyrine eliminationand clearance, galactose elimination capacity, MEGX generation fromlidocaine and methionine breath test. In a study of other patients,cholate Cl_(oral) and cholate shunt were better than caffeineelimination or antipyrine elimination and clearance in identifyingpatients at risk of future decompensation.

Combination Tests—Quantitative Tests of Liver Function (QLFTs).

Comprehensive assessment of functional hepatic reserve may require onereliable quantitative test or a combination of quantitative liverfunction tests. However, few, if any, studies have compared the resultsof more than two tests within the same cohort of patients largelybecause of the complexity of some of the tests.

In one study herein, QLFTs in HALT-C patients were examined for theutility of multiple QLFTs in predicting cirrhosis and varices. Theseanalyses indicated that cholate shunt and oral cholate clearance wereuseful and complementary to standard clinical assessment in predictionof both cirrhosis and varices. In addition, QLFTs correlated not onlywith clinical and laboratory measures of hepatic function but alsopredicted response to antiviral therapy. One advantage of these tests isthe use of a combination of quantitative tests to comprehensively definehepatic function in selected and controlled populations. These testsprovide critical information necessary for the understanding offunctional hepatic capacity and recovery for most if not all liverconditions.

One quantitative test for hepatic condition is Cholate Clearance.Clearance of cholate is dependent upon specific high-affinity transportproteins located on the sinusoidal surface of hepatocytes and isproportional to hepatic blood flow and hepatocyte function. Clearance ofcholate from portal blood or first-pass hepatic extraction, can bemeasured in humans using dual isotopes (e.g. stable isotopes) andsimultaneous oral and intravenous administration. Simultaneousadministration is defined by administration of oral and intravenous testcompounds within 5 minutes of each other. Stable (¹³C, ²H, ¹⁵N, ¹⁸O) orradioactive isotopes (¹⁴C, ³H, Tc-99m) can be used. Advantages of stableisotopes are the lack of exposure to radioactivity, natural abundance,and the specificity of the analyses used for test compoundidentification (mass determination by mass spectrometry). Cholateescaping hepatic extraction enters the systemic circulation and isdefined as the cholate shunt. In patients in the previously mentionedHALT-C trial, cholate shunt correlated with for example cirrhosis onliver biopsy, varices on endoscopy, splenomegaly on ultrasonography,platelet count (a reflection of infection), and biochemical markers ofdisease severity. In this study the method of measuring the cholateshunt required sampling of blood for at least 3 hours resulting inprolonged discomfort and delay to the patient. In one embodiment, acholate clearance test can be used alone, or in combination with otherhepatic assessment tests.

One Quantitative Liver Function Test (QLFT) is the Cholate Shunt assay.A cholate shunt assay determines a relative value for predictingclinical outcome or monitoring of hepatic disease progression. In oneembodiment of the invention, ^(n)C-cholate is administered intravenouslyand ²H4-cholate is administered orally to a subject suspected of havingor developing a liver disorder. In accordance with this embodiment,blood samples for measurement of cholate isotopes are obtained at abaseline and several times after the baseline. For example, samples maybe taken at 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120, 150 and lessthan 180 minutes post-dose where a total of 14 blood samples may becollected over 180 minutes. Alternatively, fewer samples may beobtained. In another aspect, five samples are collected at 5, 20, 45, 60and 90 minutes post-administration. In a further aspect, samples arecollected up to a half an hour after administration of cholate. Fromthese samples, intravenous and oral cholate clearance curves can begenerated. The least squares method can be used to determine the areaunder the cholate clearance curves. Next, the liver shunt fraction, anindicator of liver function, is calculated using a method described inthe Exemplary Operations section.

In order to reduce patient discomfort, time and resources, in oneembodiment a deconvolutional analysis may be used to generateintravenous and oral distinguishable agent clearance curves. To assessone or more hepatic conditions in a subject in the optimal amount ofblood draws and time, spline functions, calculated elimination rates anddirect integration of mathematical equations may be used to reduce thenumber of blood draws and reduce the time required for assessment.

Each of the above tests has certain advantages and disadvantages but fewstudies have examined the value of quantitative tests or compared therelative benefits of individual tests in either predicting diseaseprogression or in monitoring response to long-term maintenance therapy.The present invention proposes quantitative tests that may predictoutcome and therapeutic endpoints, in subjects with a liver condition(e.g., chronic hepatitis C with compensated cirrhosis).

In an earlier study, hepatic function was compared betweenChilds-Turcotte-Pugh A cirrhotics and normal controls by measuring theclearances of antipyrine, caffeine, and cholate labeled with stableisotopes, and cholate shunt. First, Childs-Turcotte-Pugh A cirrhoticswere chosen because use of QLFTs to quantify the degree of hepaticimpairment in cirrhotics with obvious clinical deterioration(Childs-Turcotte-Pugh B and C) was assumed to be of little additionalutility above standard liver tests and clinical assessment. Second, theexisting literature suggests that Childs-Turcotte-Pugh A cirrhoticslikely have a wide range of hepatic functional impairment ranging fromnearly normal to severely abnormal making this condition ideal forstudying functional differences by QLFTs. Quantitation of liver functionwithin this group might yield cutoffs for test results more likely topredict subsequent clinical outcome. Third, the use of multiple testsallowed comparison of the predictive value of a number of quantitativetests. Specifically, these tests may provide whether compounds clearedby hepatic metabolism including, but not limited to, for examplecaffeine and antipyrine or those whose clearance was flow dependentincluding but not limited to cholate, lidocaine, inderol, andnitroglycerine are informative with respect to functional reserve andrisk of decompensation.

These studies revealed that the hepatic clearances of the administeredcompounds were significantly reduced in patients with cirrhosis but therange of functional impairment overlapped into the range of healthycontrols. Five patients decompensated and required hepatic transplant ordied from liver failure. Caffeine elimination or antipyrine clearancefailed to separate these 5 patients from the cirrhotics who remainedstable.

In contrast, the clearance of orally-administered cholate and first-passelimination of cholate (cholate shunt) correlated with the patients whoultimately demonstrated evidence of decompensated liver disease duringthe follow-up period. The values for oral cholate clearance and cholateshunt in decompensated patients differed from the values measured forstable patients. These results indicated that quantitative tests, inparticular dual cholate clearance, identified Childs-Turcotte-Pugh ClassA cirrhosis patients at greatest risk for decompensation. Although thestudy focused on CTP Class A patients, the results may also be valid forpatients with more advanced disease (CTP Class B or C) especially inprediction of severe complications (ascites, variceal hemorrhage,encephalopathy), hepatoma, or need for transplantation.

Thus, in one embodiment of the present invention, patients withChilds-Turcotte-Pugh Class A (and possibly CTP class B or C) cirrhosismay be tested for hepatic health. In a more particular embodiment, thecholate shunt assay detailed herein may be used to evaluate patientswith Childs-Turcotte-Pugh Class A cirrhosis to analyze hepatic health.In another embodiment, a dual cholate clearance and shunt test may beused to evaluate patients with Childs-Turcotte-Pugh Class A cirrhosis toanalyze hepatic health. In another embodiment, a dual cholate clearanceand shunt test may be used to evaluate patients withChilds-Turcotte-Pugh Class A cirrhosis to analyze hepatic health inorder to assess the need for therapeutic intervention. Alternatively, acholate shunt assay and/or an oral cholate clearance assay may be usedto assess hepatic health of a subject undergoing a therapeutic treatmentfor a liver condition such as, but not limited to, Childs-Turcotte-PughClass A cirrhosis.

In one embodiment, quantitative testing of hepatic function is usefulfor predicting outcome in a subject with fibrotic liver diseaseotherwise clinically stable with no biochemical or clinicaldecompensation. In addition, quantitative tests are useful astherapeutic assessments in patients who have mild hepatic dysfunctionaround baseline and who achieve a positive therapeutic response. Inaddition, QLFTs may also measure rate of disease progression during thecourse of a trial where lack of response or failure to receive therapyis likely to further impair hepatic function.

Cholic acid is the primary breakdown product from cholesterol. The adultpool size is from 1 to 3 grams. Thus the doses for administrationsuggested herein: from 20 to 60 mg, increase the cholate pool size nomore than about 6%, an expansion that is unlikely to alter the normalmetabolism of cholate in the human body. Given the enterohepatic cyclingof cholate and about 5% loss with each cycle, 30 to 50% of administeredcholate is eliminated each day; within 5 days, virtually alladministered cholate is eliminated from the human body. Stableisotopically labeled compounds are commercially available. For example,¹³C- and ²H-labeled cholic acid compounds can be purchased fromSigma-Aldrich, CDN Isotopes and Cambridge Isotope Laboratories, Inc.

The selection of the correct intravenous dose of labeled cholic acid isimportant; the IV dose must be safe, not cause injection site reaction,remain in the intravascular space, and achieve physiologic blood/serumconcentrations. For accurate assessment of the cholate shunt theintravenous dose of labeled cholate must be restricted to theintravascular compartment and maintain residence in this space. Cholateis physiologically bound to albumin by interaction with specific domainswithin the albumin molecule. If cholate were injected directly into aperipheral vein, unbound to albumin, free cholate could diffuse intoextravascular spaces or injure the vein into which it is injected. Inone aspect of the invention, the labeled cholic acid intended forintravenous administration is prebound to albumin; specifically humanserum albumin. Human serum albumin is FDA approved for use in humans. Inone aspect, a dose of albumin is chosen that ensures complete binding ofthe labeled cholic acid. In one specific aspect, 5 mL of 25% human serumalbumin is prebound to 20 mg labeled cholate prior to intravenousadministration. Selection of the injection site for intravenousadministration of labeled cholate should be such that adequate veindiameter allows for successful cannulation in nearly all cases. In oneaspect, the antecubital vein is an appropriate injection site. In aspecific aspect, the 10 mL volume of the intravenous dose is easilyadministered within a one minute interval by bolus injection.

The use of albumin-bound cholate maintains cholate in the intravascularspace. Although the intravascular space includes serum and plasma;albumin is resident in serum, not plasma. By using albumin, the labeledcholate resides in serum, avoiding non-specific binding to clottingfactors. Albumin binding also avoids diffusion of cholate intocirculating cells such as red cells, WBCs, or platelets. Serum is easyto transport to a central laboratory for subsequent processing andanalysis. Further, cholate is not degraded by constituents of serum.Although freezing of the serum sample is recommended for long termstorage or transport, it is not necessary, so long as risk ofevaporation is eliminated by securely fastened tubes.

Clearance of the IV dose is related to, or represents, total hepaticblood flow. Clearance=[hepatic blood flow]×[hepatic extraction ofcholate]. Hepatic extraction of cholate=[uptake per cell]×[number ofcells].

In one embodiment, the dosage form for oral administration of thelabeled cholate is a solution form. Intestinal absorption andbioavailability of administered cholate is best achieved from a solutionform. In one embodiment, the dissolution solution for oraladministration of labeled cholate is a bicarbonate solution. Cholate iseasily dissolved in aqueous sodium bicarbonate. A solution of sodiumbicarbonate and cholate is not very palatable, so in one aspect,flavoring is used to improve acceptance of the orally administered dose.The flavoring can be natural or artificial. In a specific aspect, thesolution of labeled cholate in sodium bicarbonate is a solution of anon-citrus juice, such as apple or grape juice.

Certain methods of the invention comprise administration of isotopicallylabeled cholate. In one exemplary method about 20 mg of 24-¹³C cholicacid was mixed with 5 ml 25% human albumin and injected through anintravenous catheter over 2 min. In another exemplary method about 40 mgof 2,2,4,4-²H cholic acid was dissolved in NaHCO₃ aq. and taken orally.In one example, blood was drawn at baseline and 5, 10, 15, 20, 30, 45,60, 75, 90, 105, 120, 150 and less than 180 minutes post-dose.

In one exemplary method, the cholates are isolated by extraction fromserum with Sep-Pak C18 cartridge, acidification, ether extraction,methylation, TMS derivatization and capillary GC-MS isotope ratiometry.

The cholate shunt calculation is based on AUC_(po)/AUC_(iv) which iscorrected for administered dose-Dose i_(v)/Dose_(po). The cholate shuntis expressed as 100% to allow a range for reporting that is easy tocomprehend and relate to the provider of healthcare and the patient, andis a number that can be easily followed. In one exemplary method, theCholate shunt (%) may be calculated asAUC_(oral)/AUC_(iv)×Dose_(iv)/Dose_(oral)×100%.

Published methods for sample preparation of serum bile acids for eitherGC-MS or LC-MS are multistep, complex, and typically require severaldays. In one embodiment of the disclosure, an improved method of sampleanalysis is provided. In this embodiment sample processing for HPLC-MSis straightforward, rapid and does not require sample derivitization, incontrast to GC-MS sample preparation. In one embodiment, sodiumhydroxide is added to the serum samples, samples are subjected to solidphase extraction by passage over SepPak C18 cartridges, samples areeluted from cartridges, the samples are dried, the samples are acidifiedwith an HCl solution and extracted with ether. The ether is dried andsamples are taken into the HPLC mobile phase buffer. Samples are addedto an autosampler and subjected to HPLC-MS with selective ionmonitoring.

In one aspect, analysis of MS data for peak area determination andsubsequent calculations for Excel spreadsheet or other spreadsheet ordata analysis software can be performed by the HPLC-MS software package.In another aspect, HPLC-MS data can be automatically transferred to PCworkstations. In another aspect, conversion of peak areas to cholateconcentration is performed using standard or calibration curves. In afurther aspect, baseline measurement of natural abundance is alsoperformed. In one aspect, unlabeled cholate in excess, e.g., 3 ug/mL, isused as an internal standard for quantifying the isotopically labeledcholates. In another aspect, correction for ion overlap betweenadministered isotopes is performed using simultaneous equations forsolving minor ion bleed-over and contamination of ion peaks. In afurther aspect, determination of unlabeled cholate is performed onbaseline samples. In another aspect, quality control samples bracket thecharacteristic concentrations achieved during performance of the IV andPO clearance tests. In one embodiment, the area under the curve (AUC)for administered isotopes of cholate is performed by plotting theconcentration of the cholate in the sample versus time. In one aspect, aminimal model for accurate determination of AUC utilizes five samplecollection times over 90 minutes. In a specific aspect, the bloodsamples are collected at 5, 20, 45, 60 and 90 minutes post-dose.

In one embodiment, the data is reported to the healthcare provider inany one of several formats. In certain aspects, the data can be reportedout to the health care provider as one or more of cholate clearance fororally administered cholate, the cholate clearance for intravenouslyadministered cholate, the cholate shunt, Cholate Kelim, Cholate Vd,total hepatic blood flow, apparent shunted volume. In another aspect,data can be corrected for weight, body mass index, and ideal bodyweight. In certain aspects, the risk of varices, size of varices, riskof cirrhosis, and stage of fibrosis can also be reported.

General Considerations for the Cholate Shunt

In one embodiment, the present invention concerns detecting cholate in asample of a subject for prediction of the onset or progression of ahepatic condition.

Healthcare providers are in need of an accurate and relativelyinexpensive and easily administered test for early predictors of organfailure. In one example, a quick test for the prediction of hepatichealth is needed. In other examples, a quick, accurate, and relativelyinexpensive test for the prediction of liver failure is needed. Becauseof the nature of cholate as a predictor of hepatic health of a patientsuch as a patient with a liver condition (e.g., chronic hepatitis C), amethod that can alert a healthcare provider that hepatic health isworsening or improving with treatment would be beneficial from aclinical perspective. This information can alert the health careprovider that intervention by a therapeutic treatment may be requiredimmediately. The application of such methods is important for patientswith a propensity for organ failure such as hepatic failure, for examplein chronic hepatitis C patients. In addition, the application of suchmethods is important for patients undergoing organ transplantation suchas liver transplantation. Other situations where these techniques may beuseful include kidney, lung, heart and bone marrow transplantations. Anydisease that might alter the hepatic condition could be an indicationfor use of the test.

Methods for determination of cholate clearance in a patient aredisclosed herein. A relatively cheap, quick and reliable assay willpromote optimal application of a health provider's resources to diagnoseorgan insufficiencies such as hepatic insufficiencies and otherconditions of altered hepatic function. Alternatively, a quick andreliable assay such as methods for detection of cholate clearance in asample may be used to monitor response to drug regimen and assesstreatment efficiency, leading to a decreased loss of life and decreasedcost. These methods may be used to assess the efficiency of onetherapeutic treatment versus another or comparing various dose levels ofthe similar or different treatments on a patient suffering from ahepatic condition.

Advantages of the cholate shunt assay include reliable results thatcorrelate with organ health (e.g., liver health) and use of a naturallyoccurring substance rather than a drug in a variety of subjects bearingor predisposed to an organ condition. Because the assay utilizesaccurate and specific detection methods, the reproducibility andreliability of the test will provide accurate sample analysis. Theequipment and methodologies used to analyze the presence of cholate mayrequire chromatography (for example, gas chromatography (GC) or highpressure liquid chromatography (HPLC)) and mass spectrometry (MS) withappropriate training of the operator. However, the assay does notrequire any unusual or complex techniques outside the general spectrumof assays utilizing GC/MS or HPLC/MS technology. The assay isstraightforward since the introduced cholate is distinguishable. Theassay is sensitive and requires a short time period, typically in thetime range of 90 minutes or less. Since the cholate shunt assay can beused to measure cholate clearance early in disease progression and maybe combined with other assays, it provides more complete data thanpresently used methods for early intervention and treatment of hepaticconditions.

The evaluation of the presence of distinguishable cholate in the contextof other parameters has suggested that the cholate shunt assay issensitive to altered states of organ health, including liver incritically ill patients.

Because of the vital importance of earlier targeting of therapies in ashorter amount of time, many markers have been explored for earlydiagnosis of hepatic disease or condition. An assay requiring 3 hours orlonger causes increased discomfort to a subject undergoing such a test.

The cholate shunt assay may be utilized to assess liver function in apatient. In some embodiments, cholate shunt assay results may beanalyzed in an individual having or predisposed to a liver condition.Non-limiting examples of liver conditions include but are not limited tocirrhosis, splenomegly, varices, cancer and chronic hepatitis Cinfection.

In another embodiment, cholate shunt assay results may be analyzed in anindividual undergoing an organ transplant. Non-limiting examples oforgan transplants include but are not limited to liver transplantrejection, delayed function of the liver transplant, recurrent diseasein the transplanted graft, and liver injury.

In yet another embodiment, the cholate shunt assay may be used toanalyze healthy subjects to assess organ health in steady state and intimes of altered (pathologic or physiologic) conditions, including thespecial physiologic states of organ transplant.

Evaluation and monitoring of the clearance of cholate may be used toassess liver function in a patient. Whether or not organ (or cellular)destruction can be minimized after events such as organ injury orprolonged exposure to an infection (e.g., Hepatitis C) may depend, inpart, upon the early introduction of therapeutically relevanttreatments. In order to eliminate, minimize or attenuate suchdestruction in an individual who has undergone or is undergoing organdamage, failure or similar event, it would be helpful to predict theseevents earlier in progression rather than later. By comparing theindividual's specific level of clearance of cholate to a normal healthycontrol, or within a given individual over time, a treating physicianmight determine whether the subject needs to be treated immediately orotherwise observed for a period of time.

Under conditions when cholate clearance is detectably altered in asample of a subject, such as after organ injury, organ transplant orprolonged infection, it becomes critical that the treating healthcareprovider has reliable information available about an individual'sconcentration of cholate in the sample. For example, a relatively highconcentration of the orally administered cholate in the blood isespecially likely to occur when the subject is undergoing a delayedliver transplant graft function. In addition, a relatively highconcentration of orally administered cholate in the blood is especiallylikely to occur when a subject with a liver condition (e.g., HepatitisC) has experienced hepatic insult. Thus, when a patient's organ activitysuch as hepatic activity is impaired as indicated in the examples above,a healthcare professional may intervene and administer a therapeutictreatment to attenuate the condition or possibly reverse failure of theorgan. These interventions may avoid permanent damage or death of thepatient. In addition, a healthcare professional may monitor thetherapeutic treatment of the subject by obtaining samples from thepatient after treatment and analyzing the presence of cholate in thesample and assessing the condition of the patient based on thesefindings. Therapeutic treatments may be altered depending on the changein cholate detection or concentration of cholate present in the sample.

Healthcare professionals have been hindered by an inability to prescribeindividualized doses of agents tailored to the unique physiologicalresponses of a particular subject early enough in the process of organfailure. In the absence of such data, most treatments are introduced toa patient too late. Early diagnosis and intervention with a treatmentsuch as introduction of fluids, sodium bicarbonate, atrial natriureticpeptides, growth factors, dialysis, or any therapy for prevention oforgan failure may either attenuate the progression of the condition oralleviate the symptoms of the condition. Thus, a rapid test to assessthe onset of organ failure would be extremely useful for diagnosis andtherapeutic monitoring.

In one embodiment of the disclosure, hepatic health of a subject may bemonitored using either, or both of, the cholate oral clearance assay andthe cholate shunt assay to assess hepatic function. In anotherembodiment either or both of the cholate oral clearance assay or thecholate shunt assay may be used in combination with one or more otherQLFTs disclosed herein to assess hepatic function. In accordance withthese embodiments, therapeutic intervention may be administered to thesubject as necessary. In another embodiment, hepatic health of a subjectundergoing therapeutic intervention may be assessed using either, orboth of, the cholate oral clearance assay and the cholate shunt assay toassess hepatic function.

In one embodiment, a cholate clearance assay or cholate shunt assay maybe used in combination with one or more other hepatic assessment tests.Example protocols for additional quantitative testing include, but arenot limited to, additional tests discussed below.

In one embodiment, for example, participants can undergo quantitativeassessment of hepatic functional reserve at baseline, with follow-up at2 and 4 years of the maintenance treatment protocol. At each time point,quantitative testing can be performed after 3 days of a caffeine-freediet and an overnight fast. Patients can report to their respectivetreatment centers and be admitted to the respective clinical researchcenter. An indwelling catheter will be placed in an antecubital vein andbaseline blood drawn. In one aspect, test compounds can be administeredboth orally (e.g, ²H4-cholate, caffeine, antipyrine) and intravenously(e.g., ¹³C-cholate, galactose, lidocaine). In another aspect, testcompounds can be administered orally only (e.g., ²H4-cholate).

Intravenous ¹³C-cholate, 20 mg, is dissolved in NaHCO₃ solution, passedthrough a micropore filter, and placed in sterile, capped glass vialsprior to use. This preparation can be mixed with 5 ml of 25% humanalbumin solution just prior to intravenous injection. In one example,blood samples for measurement of cholate isotopes can be obtained atbaseline and 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120, 150, and lessthan 180 minutes post-dose (14 samples, 7 ml red top tubes). In anotherexemplary method, blood samples for measurement of cholate isotopes canbe obtained at 5, 20, 45, 60, and 90 minutes post-dose (5 samples).

In one example, a Galactose 30% solution, 100 ml, is given intravenouslyover 5 minutes. Blood is obtained at 20, 40, 60, and 80 minutespost-dose (7 ml gray top tubes). Samples must be kept on ice orrefrigerated. Spin samples for 10 min at 3000 rpm, remove plasma andkeep at −20° C. until analysis. “High-dose” samples are diluted 1:2 inMilli-Q water before testing.

The standard test dose for intravenous infusion of lidocaine in the MEGX(monoethylglycine zylidide) assay is 1 mg/kg over 2 minutes. However arecent report suggested that a lower dose (less than 1 mg/kg such as 0.5mg/kg) may be better tolerated, associated with fewer side effects (30vs. 53%, sensory symptoms), and gives similar accuracy in quantitatinghepatic function. In this experimental example 0.5 mg/kg dose will beused. Blood is obtained at baseline and 15 minutes post-infusion.Results are reported as the difference between the concentrations ofMEGX at 15 minutes post-lidocaine, compared to concentration atbaseline.

In one exemplary method, saliva samples, for measurement of antipyrineand caffeine, will be obtained at baseline and at 6, 12, 24, 36, 48, and60 hours post-dosing (7 samples, 5 mls each).

Other quantitative tests include Antipyrine and Standard Caffeine Test(saliva). In one exemplary method, salivary samples are centrifuged toremove particulates, dispensed into 1 ml aliquots for analysis, andinternal standard (phenacetin) added. After extraction with organicsolvent, samples can be quantitated using HPLC (for example, with a WISPsystem). Kinetic parameters (k_(elim), V_(d), Cl) can be calculated fromthe plot of salivary concentration vs. time. Concentrations ofantipyrine in saliva are equivalent to that found in plasma and allkinetic parameters for antipyrine can be determined from saliva.K_(elim) is equivalent from saliva and plasma. In contrast, albuminbinding of caffeine reduces the diffusion of caffeine into saliva andcaffeine concentrations are, therefore, lower in saliva. This effect canlead to falsely high V_(d) and apparent clearances for salivary caffeinewhen compared to the same parameters determined from serum samples. Newand improved methods for assessing caffeine clearance might bebeneficial. K_(elim) from the caffeine data and k_(elim), V_(d), and Clfrom the antipyrine data can also be used.

In one embodiment, the hepatic condition of a subject may be assessedusing a test which utilizes an agent labeled by two or more differentdistinguishable agents, for example, to assess liver metabolism. Thesedistinguishable agents may be introduced to a subject at different timesand different dosages and metabolically tracked in the subject. Inaccordance with this embodiment, the distinguishable agents may includedifferent distinguishable isotopes (e.g. stable isotopes: ¹³C, ²H, ¹⁵N¹⁸O or radioactive isotopes ¹⁴C, ³H) linked to for example, an agentreadily metabolized by the liver such as caffeine. Distinguishablecaffeine can be purchased (for example CDN Isotopes Inc., Quebec, CA).This test is referred to as a multi-isotope caffeine metabolism test. Toassess hepatic condition in a subject, distinguishable caffeine may beintroduced orally and/or by IV and introduced to a subject over a periodof time. After introduction to the subject, distinguishable caffeinemetabolites are tracked by assessing saliva and/or blood samples. In oneembodiment, hepatic condition of a subject may be assessed using 3different isotopically distinguishable caffeine solution (triple isotopemethod: TIME) introduced to a patient and sometime later obtainingsaliva and/or blood samples where metabolism of the solution isindicative of the subject's hepatic condition. It is contemplated thatthe time of administration of the distinguishable agent may vary from asshort as a few hours to as many as 36 hours before a sample is obtainedand metabolism assessed. In one particular embodiment, eachdistinguishable caffeine solution may be introduced to a subject at adifferent time and one saliva or one blood sample obtained from thesubject sometime after administration of all caffeine solutions to thesubject.

In another embodiment, hepatic condition of a subject may be assessedusing a test including caffeine labeled by two or more distinguishableagents, introduced to a subject and metabolically tracked in the subjectin combination with another hepatic assessment test such as a hepaticblood flow test. For example the multi-isotope caffeine metabolism test(e.g. triple isotope method) may be combined with a cholate clearance orcholate shunt assay disclosed herein. Other tests may be combined withthe multi-isotope caffeine metabolism test such as other metabolism orhepatic blood flow tests that reflect hepatic condition. Outcomes ofthese tests are indicative of hepatic condition and thus assessment ofcurrent or future need of treatment to alleviate any hepatic conditionin a subject may be recognized. In addition, any methods disclosedherein may be used to assess hepatic condition in a subject undergoing atreatment for a condition. If required, a treatment of such as subjectmay be modified in accordance with the hepatic condition.

In the present invention, one advantage of using a multi-isotopecaffeine test is that dietary caffeine will not interfere with theassay. In addition the data obtained from elimination of caffeine froman individual is near total elimination of the caffeine. The samplingpost administration of the distinguishable solution may be a single timepoint.

Unlike the typical clearance, metabolism, or breath test analyses ofcaffeine, caffeine tests disclosed herein avoid caffeine interference bydiet or drug caffeine. In addition, caffeine tests of the presentinvention assess a more global assessment of caffeine metabolismcompared to traditional caffeine breath tests that assess a singlepathway.

In one embodiment, the predictive value of the quantitative tests can beestablished as follows. The results of the baseline studies can becharacterized by one or more of a mean, median, distribution, andconfidence intervals (CIs) for one or more of the measures of hepaticfunction (caffeine k_(elim), antipyrine k_(elim), antipyrine Vd,antipyrine clearance, galactose elimination capacity, MEGX15 min,cholate k_(elim) iv, cholate Vd iv, cholate Cliv, cholate Clpo, cholateSF, and perfused hepatic mass). The median value for each test may beused to divide the patient sample into two groups for analysis of theability of the test to predict clinical progression. The composition ofthe groups will change for each test analyzed based upon the baselineresults for the specific test undergoing evaluation. For example, themedian value for caffeine k_(elim) may be 0.04 h⁻¹. Values below 0.04h⁻¹ indicate poorer function and greater likelihood for early clinicaldecompensation. In one aspect, the median value for cholate SF is 30%;values above 30% indicate poorer first-pass clearance and greaterlikelihood for early decompensation. For example, Patient A tests mayindicate caffeine k_(elim) 0.06 h⁻¹ and cholate SF 55%. In the analysisof the predictive value of these tests, Patient A's long-term outcomewould be analyzed with the caffeine group likely to have a betteroutcome but with the cholate group likely to have a poorer outcome.Predictive value is calculated by standard technique using 2×2 tablesthat define true positives (TP) and negatives (TN) and false positives(FP) and negatives (FN). A hypothetical analysis is shown in Table 1 forthe example of results from cholate SF testing.

TABLE 1 Predictive value of cholate shunt fraction in hepatic diseaseprogression. Disease Progression Cholate SF > 30% Cholate SF < 30% YesTP FN No FP TN Positive Predictive Value = TP/[TP + FP] × 100% NegativePredictive Value = TN/[TN + FN] × 100%

The predictive value of the various tests may be compared andinteraction between the quantitative tests in predicting outcome will beperformed by multivariate analysis of the continuous independentvariables (quantitative tests) against the binomial dependent variable(development or absence of clinical decompensation).

In one embodiment, the quantitative tests can be used to determine anoutcome or endpoint of therapy. The control group of a given study mayexperience progressive decline in hepatic function as measured byquantitative tests. Each patient may serve as his own control; testresults in years 2 and 4 of treatment will be subtracted from baselinetest values. The absolute and percent change from baseline will bedetermined for each patient at each time point and mean, median,distribution, and confidence intervals determined. Statisticalsignificance of differences in the changes from baseline betweentreatment and control groups may be determined by ANOVA. In addition,changes in quantitative tests will also be compared to changes infibrosis scores, fibrosis morphometry, standard biochemical tests, andconcentrations of HCV RNA. Kaplan-Meier curves and Log-Rank tests(nonparametric) will also be used to compare the changes in quantitativetests between the two patient groups.

Other tests may be utilized for combination analysis such as, forexample, sulfur colloid distribution parameters. In one exemplarymethod, the cholate shunt or cholate clearance assay may be combinedwith analysis of sulfur colloid distribution. The distribution of sulfurcolloid from the planer scan can be assessed by any means known in theart. In another example, distribution of sulfur colloid between liverand bone marrow may be assessed by any means known in the art and usedin combination with any assay disclosed herein.

In one embodiment, distinguishable compounds, agents or solutions usedherein include compounds that are traceable or trackable. Thesecompounds linked to an agent of interest (e.g., cholate or caffeine) maybe followed as they are processed or passed through a subject before,during and/or after administration of the distinguishable agent to thesubject. Cholates used in any one of these assays might be labeled witheither stable (¹³C, ²H, ¹⁸O) or radioactive (¹⁴C, ³H) isotopes. Thesesame isotopes and potentially ¹³N or Tc-99m could be linked to any ofthe other agents described and referenced herein. A number of other testcompounds are listed in the descriptions above and could be used aspotential substitutes for either cholate (other bile acids, propranolol,lidocaine, nitroglycerin) or caffeine (antipyrine, erythromycin,lidocaine-MEGX, midazolam, dextromethorphan, and any other xenobiotic orcompound metabolized by the P450 system).

A radionuclide may be bound to an agent such as cholate either directlyor indirectly by using for example an intermediary functional group.Intermediary functional groups which may be used to bind radioisotopeswhich exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetraceticacid (EDTA). Examples of metallic ions suitable for use in thisinvention are 99 mTc, ¹²³I, ¹³¹I ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I,⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

In accordance with these embodiments, agent(s) thereof may be labeled byany of several techniques known to the art. The methods of the presentinvention may also use paramagnetic isotopes for purposes of in vivodetection. Elements particularly useful in Magnetic Resonance Imaging(“MRI”) include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe.

Other labeled compounds are contemplated such as fluorescent compounds.As known in the art any distinguishable component may be covalentlylinked or attached in any manner to the agent for detection in a samplesuch as fluorescent dye etc.

After hepatic condition of a subject has been assessed by one or more ofthe QLFTs disclosed herein, it may be determined that a therapeutictreatment is necessary for the subject. Likely treatments orinterventions in hepatic conditions include but are not limited to aninterferon, e.g., interferon alpha-2b, peginterferon; ribavirin; acombination of an interferon and ribavirin; any new and emergingtreatments for either or both hepatitis B and C; lamivudine, adefovir;tenofovir; telbivudine; telaprevir; boceprevir; ursodeoxycholic acid;treatments for NASH, TIPS; hepatic resection; and hepatictransplantation.

In one aspect, one or more of the QLFTs disclosed herein may be used tomonitor treatment and/or disease progression.

In still further embodiments, the present invention discloses kits foruse with the methods and comparison methods described herein. One ormore distinguishable agent(s) provided in a kit may be employed toassess organ health in a health facility and/or a home kit format.Distinguishable agent(s) such as a hepatic blood flow assessing agentand/or hepatic metabolism assessing agent (e.g. cholate and/or caffeine,respectively) may thus comprise, a suitable container means, an oraldose of distinguishable agent to possibly be administered outside of ahospital environment. In addition, a second IV dose may be administeredin a healthcare facility. Sample tubes for collection of the bodilyfluid samples such as blood or saliva for collection either inside oroutside a healthcare facility may also be included. In one example, akit may comprise an oral and an IV dose of one or more distinguishableagents and sample tubes for collection of samples over a period of lessthan 3 hours after administration of the distinguishable agents. Inanother example, a kit may comprise an oral dose of one or moredistinguishable agents and sample tubes for collection of samples over aperiod of less than 3 hours, after administration of the distinguishableagents. In another example, a kit may comprise components necessary fora test period of 90 minutes post administration of one or moredistinguishable agents. In a further example, a kit may comprisecomponents necessary for a test period of 30 minutes post administrationof distinguishable agents.

Another kit may include distinguishable metabolic indicators of hepatichealth such as distinguishable caffeine. It is also contemplated that acombination kit having both a metabolic indicator such as caffeine and ahepatic blood flow indicator such as cholate may be useful to assessoverall hepatic health of a subject.

Further suitable reagents for use in the present kits include thetwo-component reagent that comprises a distinguishable agent detectionsystem and a metabolic function detection system. The kits may furthercomprise a suitably aliquoted composition of the specific agent such ascholate, whether labeled or unlabeled, as may be used to prepare astandard curve for a detection assay.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the distinguishable agent may be placed, and preferably, suitablyaliquoted. The kits of the present invention will also typically includea means for containing the distinguishable agent and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained. In addition, the kits may contain a productfor diluting the distinguishable oral agent such as a fruit juice orother liquid.

In certain embodiments, determination of oral cholate clearance, withoutsimultaneous determination of intravenously administered cholateclearance, is disclosed. Unique uses of oral cholate clearance includesituations where avoidance of an intravenous dose of isotopicallylabeled cholate is desirable; such as in, for example, pediatricpopulations. Use of the oral cholate clearance test (cholate clearancepo) is a robust predictor of clinical outcome, as shown in Example 13.

Clearance of orally administered labeled cholate and cholate shunt canbe useful in monitoring patients with liver disease. Certain embodimentspertain to specific clinical applications of the oral cholate clearance(Cholate Cl_(oral); Cholate Clearance po; HepQuant-Oral) and/or cholateshunt (HepQuant-Dual) tests wherein results are correlated with diseaseseverity.

In certain aspects, the methods of the disclosure, for example, oralcholate clearance and the cholate shunt, can be used for selection ofpatients with significant fibrosis, and cirrhosis; selection of patientsfor endoscopy who are likely to have varices; selection of patients forendoscopy with large varices (varices at risk to bleed); identificationof patients with chronic hepatitis C who are unable to respond topeginterferon/ribaviron; tracking hepatic improvement in patients whorespond to therapy; and identifying the subgroup of patients who arelikely to experience future decompensation. Clinically relevant valuesfor various conditions are shown in Table 2.

TABLE 2 Clinical Applications for Oral Cholate Clearance and CholateShunt. Table 2. Clinical Applications for Oral Cholate Clearance andStat Test, Stat Test, Cholate Shunt. HepQuant- Score, and Score, andClinical Application Oral P value HepQuant-Dual P value ReferenceSelection of patients Cl < 1250 mL/min, regression Shunt > 30%, atregression Aliment with significant at analysis, this value of analysis,Pharmacol fibrosis this value of r = −0.52, shunt the r = 0.49, Ther Clthe P < 0.001 calculated Ishak P < 0.001 2008; 27: 798-809 calculatedfibrosis score Ishak fibrosis from the score from the regressionregression equation was 2.6 equation was 2.6 Selection of patients PPVof ROC PPV of Shunt > ROC Aliment with cirrhosis Cl < 1250 mL/min:analysis, c- 30%: 51% analysis, Pharmacol 53% stat, PPV of Shunt >c-stat, Ther PPV of P < 0.0001 50%: 72% P < 0.0001 2008: 27: 798-809 Cl< 750 mL/min: 69% Selecting patients for PPV of ROC PPV of Shunt > ROCAliment endoscopy who are Cl < 1250 mL/min: analysis, 30%: 41% analysis,Pharmacol likely to have varices 43% c-stat, PPV of Shunt > c-stat, TherPPV of P < 0.0001 50%: 57% P < 0.0001 2008; 27: 798-809. Cl < 750mL/min: 58% Selecting patients for 100% had Sensitivity 100% had Shunt >Sensitivity Aliment endoscopy who are Cl < 1250 mL/min analysis 30%analsysis Pharmacol likely to have large 91% had 91% had shunt > Thervarices (varices at-risk Cl < 750 mL/min 35% 2007; 26: 401-410. tobleed) Identifying patients Cl < 771 mL/min, Quartile Shunt > 48%,Quartile Aliment with chronic hepatitis below this analysis above thisvalue analysis Pharmacol C who are unable to value of Cl of shunt only2% Ther respond to only 2% of of patients 2009; 29: 589-601.peginterferon/ribavirin patients achieved SVR achieved SVR Trackinghepatic Improved by Paired Improved by Paired Aliment improvement in 32%with analysis, 25% with SVR analysis, Pharmacol patients who respond SVRand P < 0.0001 and no change in P = 0.003 Ther to therapy worsened bynonresponders 2009; 29: 589-601. 10% in nonresponders Identifying theOutcomes by Kaplan- Outcomes by Kaplan- Hepatology subgroup of patientstertiles: Meier tertiles: Meier October who are likely to Cl < 9.5mL/min - And ROC Shunt > 45% - And ROC Issue experience future 47% ofthese analysis, all 42% of these analysis, all Accepted fordecompensation patients had P values < patients had P values <presentation outcomes; 0.0001. outcomes; 0.0001. at AASLD 9.5 < Cl <14.5 - Hazard 45% > Shunt > 32% - Hazard 2009 9% of these ratio for 14%of these ratio for patients had interquartile patients had interquartileoutcomes; increase in outcomes; increase in Cl > 14.5 - 5% risk of Shunt< 32% - 5% risk of of these outcome of these patients outcome patientshad was 4.37 in had outcomes. was 3.26 in outcomes. univariate By ROC,using univariate By ROC, and 3.23 in cutoff and 2.35 in using cutofftrivariate Shunt = 39%: trivariate Cl = 11 mL/kg/min: analysis 38.8%with analysis 41.5% with (included shunt > 39% had (included Cl < 11 hadcovariates outcomes covariates outcomes of biopsy compared to only ofbiopsy compared to cirrhosis 6.6% in those cirrhosis only 6.9% in andplatelet with Shunt < 39% and platelet those with count) count) Cl > 11

In further aspects, it is contemplated that the methods of thedisclosure, including oral cholate clearance and cholate shunt, can beutilized for a number of clinical applications, for example, selectionof patients with chronic hepatitis B who should receive antiviraltherapy; assessing the risk of hepatic decompensation in patients withhepatocellular carcinoma (HCC) being evaluated for hepatic resection;identifying a subgroup of patients on waiting list with low MELD (Modelfor End-stage Liver Disease score) who are at-risk for dying whilewaiting for an organ donor; as an endpoint in clinical trials; replacingliver biopsy in pediatric populations; tracking of allograft function;measuring return of function in living donors; and measuring functionalimpairment in cholestatic liver disease (PSC, Primary SclerosingCholangitis).

In a specific aspect, oral cholate clearance or cholate shunt assays canbe used repeatedly over time as a predictor of clinical outcome as shownin FIGS. 12 and 14.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. The following examples are included to demonstratepreferred embodiments.

Example 1. Procedure for Performance of Multiple Quantitative LiverFunction Tests (QLFTs)

Supplies

IV Test Compounds:

IV Solution B—30% Galactose (e.g. Pfanstiehl Laboratories).

IV Solution C—¹³C-Cholate (20 mg) (e.g. CDN Isotopes).

IV Test Compound supplied ready to use in Test Kit.

IV Solution A—2% Lidocaine (e.g. Abbott Laboratories).

PO (Per Oral) Test Compounds:

²H4-Cholate (40 mg) (e.g. CDN Isotopes).

Caffeine (300 mg) (e.g. Ruger) Antipyrine (500 mg) (e.g. Ruger).

Sodium bicarbonate (e.g. 600 mg).

Patient Testing Supplies:

25% Human Albumin for injection (5 mls) to be added to ¹³C-Cholatesolution.

Serum/plasma transfer tubes and labels.

Saliva collection tubes and labels.

IV supplies, including 250 mls NS, indwelling catheter, 3-way stopcock.

3 cc, 5 cc, 10 cc, and 50 cc syringes for administering IV testcompounds and drawing blood samples.

7 cc red top and 7 cc gray top vacutainer tubes for serum samplecollections.

Needle discard bucket.

A drinking substance such as apple or grape juice for diluting oral testcompounds.

One standard caffeine-free meal with one can Ensure® for Liver-SpleenScan.

Example Patient Preparation

Ascertain whether patient has history of allergic reactions to localanesthetics (such as at the dentist), or history of cardiac arrhythmias;if so, do not administer lidocaine. Patient is caffeine-free for 72hours prior to test day and for the subsequent 3 days of salivacollections. Patient is NPO except water after MN the night before testday. Patient has IV with 3-way stopcock and NS TKO placed before testbegins.

Exemplary Test Compound Preparation

One exemplary solution of an oral composition may contain ²H4-Cholate,Caffeine, Antipyrine, and Sodium bicarbonate (e.g. 40 mg. 300 mg, 500and 600 mg respectively). In one exemplary method, the day before thetest, water can be added to about the 10 cc mark on a tube containingthe oral test compounds to obtain the Oral Test Solution. Cap tubetightly and shake to mix. Swirl contents to get all the powder granulesdown into the water.

On the test day pour dissolved Oral Test Solution into a container suchas a urine cup. Rinse tube into urine cup with about 10 mls water. Priorto beginning the test, add a diluting liquid such as grape or applejuice (not citrus juice) to about the 40 ml mark on the urine cupcontaining the Oral Test Solution. Swirl gently to mix; do not shake orstir, or mixture may foam out of container. Have extra juice on hand forrinse.

Preparation of IV Solutions

IV Solution A (2% Lidocaine). 2% Lidocaine in a pre-packaged single-use5 cc syringe as part of the Test Kit may be provided. Test dose is 0.5mg Lidocaine/kg. Calculate appropriate dose of Lidocaine. Divide thepatient's weight in pounds by 2.2 to get kilograms; i.e., 150lbs/2.2=68.2 kg. Multiply the weight in kg by 0.5 mg/kg to get theLidocaine dose; i.e., 68.2 kg.times.0.5=34.3 mg. Divide the desired mgby 20 (concentration of 2% Lidocaine in mg/ml) to get cc's; i.e., 34.3mg/20=1.71 cc. Expel excess Lidocaine from the 5 cc syringe so that itcontains the correct dose.

IV Solution B (100 cc 30% Galactose). Galactose is prepared inindividual doses for IV. A preparation procedure may be provided. Testdose is 30 gm Galactose, or 100 mls of 30% Galactose solution.

IV Solution C (20 mg ¹³C-Cholate in 5 cc 1 mEq/ml Sodium Bicarbonate+5cc 25% Human Albumin). ¹³C-Cholate can be prepared in individual 5 ccdoses for IV. A preparation procedure may be provided. Test dose is 20mg ¹³C-Cholate (in 10 cc diluent). If vial is frozen, allow to thawcompletely before continuing. Just prior to beginning test, mix¹³C-Cholate solution with albumin as follows (this method prevents lossof test compound during mixing process). Draw up all of ¹³C-Cholatesolution (about 5 cc) in a 10 cc syringe. Draw up 5 cc albumin inanother 10 cc syringe. Inject this gently (to prevent foaming) intoempty ¹³C-Cholate vial, invert vial to rinse, then withdraw all of thealbumin back into same syringe. This rinses all of the ¹³C-Cholate outof the vial. Detach needle from the ¹³C-Cholate syringe and attach a3-way stopcock. Detach needle from albumin syringe and inject albuminthrough stopcock into ¹³C-Cholate syringe. Draw a little air into bileacid/albumin syringe and mix solutions gently by inverting syringeseveral times. Expel air.

Testing Procedure

In one exemplary method the following procedure will be used. Collectbaseline saliva and serum samples (see Sample Collection) before testcompounds are administered.

Administration of Test Compounds.

Start timer. Record 24-hour clock time as T=0.0 to 2 minutes—using 3-waystopcock, administer IV Solution A (1 mg/kg 2% Lidocaine) IV push.Record timer time −2 to 3 minutes-allow NS to flush line for 1 minute-3to 8 minutes-using 3-way stopcock. Administer IV Solution B (100 mlbolus 30% Galactose) IV push. Record timer time-8 to 9 minutes-allow NSto flush line for 1 minute-8 to 9 minutes. While line is flushing, havepatient drink oral solution of test compounds and juice. Rinse cup witha little more juice and have patient drink rinse-9 to 10 minutes. Using3-way stopcock, administer IV Solution C (20 mg Bile Acid in 5 mls 1mEq/ml Sodium Bicarbonate+5 mls 25% Human Albumin) IV push. Record timertime.

Sample Collection

Blood

¹³C-Cholate Clearance (IV Solution C). Collect all samples via the 3-waystopcock with 0.5 ml discard before each sample to prevent dilution orcross-contamination of samples. Collect 7 ml at each time point incolored tubes such as red tops for ¹³C-Cholate Clearance (IV Solution C)at the following times (time after administration/timer time): baseline(before test compounds administered), 5/15, 10/20, 15/25, 20/30, 30/40,45/55, 60/70, 75/85, 90/100, 105/115, 120/130, 150/160, and 180/190minutes.

Galactose Clearance (IV Solution B). Collect 7 ml in a different coloredcap tube like gray tops for Galactose Clearance (IV Solution B) at thefollowing times, also using same timer started at T=0 (time afteradministration/timer time): baseline (before test compoundsadministered), 20/28, 40/48, 60/68, and 80/88 minutes.

Lidocaine (IV Solution A). Collect 10 ml red tops for MEGX Concentration(Lidocaine IV Solution A) at the following times (time afteradministration/timer time): baseline (before test compoundsadministered), 15/17, and 30/32 minutes. Keep gray top tubes on ice orrefrigerated. Allow red tops to clot at room temperature for at least 30minutes. Spin all samples for 15 minutes. Transfer plasma (gray)/serum(red) to appropriate labeled tubes and freeze at −20° C. until shipping.Ship frozen.

Saliva

Have patient rinse mouth with water before each sample collection, thenstimulate saliva production by chewing parafilm squares. Collect 5 ccsaliva (foam does not count) by spitting into labeled collection tube.Collect at the following times: baseline, and 6, 12, 24, 36, 48, and 60hours. Patient may collect samples at home for convenience. If so,instruct patient regarding saliva collections at home, freezing at home,and returning samples to site. Give patient supplies for homecollection. Cap tubes tightly and freeze at −20° C. until shipping. Shipfrozen.

Liver/Spleen Scan

After completion of the blood sample collections (T=190) and 1 hourbefore Liver-Spleen Scan, give subject standard, caffeine-free meal.

Example 2. Cholate Shunt (CAshunt) Testing Procedure Utilizing GC/MSAnalysis

In one exemplary method, two hundred eighty five patients were enrolledin a clinical trial (called the HALT-C trial; Hepatitis AntiviralLong-Term Treatment to Prevent Cirrhosis Trial) and participated in aQLFT (quantitative liver function test) ancillary study. Seventy threepatients were studied twice.

Example Patient Protocol: 20 mg of 24-¹³C cholic acid was dissolved inNaHCO₃, mixed with 5 ml 25% human albumin solution and injected throughan indwelling intravenous catheter over 2 minutes. 40 mg of 2,2,4,4-²Hcholic acid was dissolved in water, mixed in juice and taken orallysimultaneously with the intravenous injection. Blood samples were drawnthrough the indwelling catheter and taken prior to isotopeadministration and 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120, 150 and180 minutes post-dose to obtain oral and intravenous cholic acidclearance curves.

Sample Preparation Protocol for GC/MS:

In this trial, patient samples were processed using the followingprotocol. In one example, dispense 0.5 ml patient serum and add 50 ul ofcholic acid standard, set aside two cholic acid controls. To each tubeadd 0.5 ml distilled water and 0.5 ml 0.02 N NaOH. Mix and incubate in a60-degree water bath for 30 minutes. Prepare Bond Elute paks (C18-0H) bywashing with 5 mls methanol and 10 mls water. Add patient sample to pak.Wash paks with 5 mls distilled water, 5 mls 13% methanol and 5 mls 87%methanol. Dry sample completely. Add 1.5 ml water to dried residue, 1drop HCl and 2 ml of diethyl ether. Vortex for 30 seconds. Centrifugefor 5 minutes to clarify layers. Collect ether layer in small,screw-capped, silanized test tubes. Repeat this step. Evaporate ether in30-degree water bath under stream of nitrogen. Methylate samples byadding 1 ml methanol, 1 ml DMP and 1 drop HCl and incubate at roomtemperature in the dark for 30 minutes. Evaporate solvent at 40 degreesin water bath under a stream of nitrogen. Make trimethylsilyl etherderivatives of bile acids by adding 0.2 ml pyridine, 8 drops HMDS(hexamethyldisilazane) and 4 drops TMCS (trimethylchlorosilane) andincubate 55-60 degrees for 2 hours. Evaporate solvents under nitrogenstream. Add 2 ml hexane. Centrifuge for 5 minutes and pour off hexane.Repeat this step. Evaporate solvent and reconstitute with 4 dropshexane. Vortex and sonicate, then transfer to injection vials. Injectonto GC/MS 6890/5973 using method Cholic2.m. This method instructs themass spectrometer to analyze each sample injection searching for ionsassociated with derivatized cholate isotopes.

Results

The full range and boundaries for quartiles of results for cholateclearances and shunt in the 282 patients at baseline, prior to entryinto the trial, are shown in Table 3. Clearances progressively declinedand shunt progressively increased as results ranged from ‘best’ to‘worst’. Cholate shunts spanned the entire range of expected result,from the low end of the normal range, 10% (‘best’), to nearly completeshunting, 91% (‘worst’).

TABLE 3 Range of study results for cholate clearances and cholate shuntin study patients. Boundaries for quartiles of test results Best 25th50th 75th Worst Cholate Cliv (mL/min) 903 457 367 305 155 Cholate Cloral(mL/min) 3036 1427 1087 768 255 Cholate shunt (%) 10 27 36 48 91

Results in 32 healthy controls were (mean+/−s.d., range): Cl_(iv)390+/−136, 155-873 mL/min; Cl_(oral) 2173+/−677, 1369-3856 mL/min; andshunt 18.5+/−5.5, 8.0-28.5%. The range of HALT-C patients (Table 3)completely overlapped with Cl_(iv) for these healthy controls. Incontrast, approximately 70% of HALT-C patients exceeded the normallimits for Cl_(oral) of 1300 mL/min and shunt of 30% (Table 3).Consequently, both cholate Cl_(oral) and cholate shunt are useful fordefining risk of cirrhosis and varices.

Example 3. Statistical Analysis

In one example, the following analysis was performed to determine ifexogenously ingested and iv administered distinguishable agents can beutilized as markers for hepatic conditions; not simply an affirmative ornegative test for hepatic conditions.

Example Study

In one example, 7 QLFTs were used to define hepatic impairment inpatients with chronic hepatitis C and bridging fibrosis or compensatedcirrhosis enrolled in the Hepatitis Antiviral Long-Term Treatment toPrevent Cirrhosis Trial (HALT C). Test results were compared with orwithout biopsy-proven cirrhosis, splenomegaly on ultrasonography, andvarices at endoscopy.

In one example study the mean age of the 248 enrolled patients was49.9+7.3 yr and 75% were male. Mean BMI (body mass index) was 29.6+5.3,40% had cirrhosis, 60% had bridging fibrosis, 93% were infected with HCVgenotype 1, and mean serum HCV RNA was 4.39+4.66×106 copies/ml. 30% hadplatelet count <140,000/ul, 25% had albumin <3.5 g/dl, 25% had INR>1.1(international normalization ratio prothrombin), 10% had bilirubin >1.2mg/dl, and 25% had AST:ALT>1 (serum aspartate transaminase: serumalanine transaminase).

In accordance with this example: ¹³C-methionine (MBT), caffeine (Caf),antipyrine (AP), and 2,2,4,4-²H-cholate (CA) were taken orally and24-¹³C-cholate, galactose (Gal), and lidocaine were administeredintravenously. These compounds or their metabolites were measured fromtimed serial samples of blood, saliva, and breath using standardtechniques. Elimination rate (kelim), volume of distribution (Vd),clearance (Cl), elimination capacity (Elim), and shunt were calculatedfrom measured analytes. Perfused hepatic mass (PHM) was determined fromSPECT liver scan. Mean test results were compared by T statistic andarea under the receiver operator curve (ROC) by C statistic. Tableresults are ordered by T statistic for association with cirrhosis. PHMhad the highest area under ROC with cirrhosis (C statistic 0.87),splenomegaly (C statistic 0.75), and varices (C statistic 0.832) andcorrelated best with platelet count, bilirubin, prothrombin time, andalbumin.

The outcome of the exemplary process was that QLFTs uncover hepaticimpairment in a high proportion of fibrotic patients with chronichepatitis C. Some of the tests, particularly CA Cloral, PHM, andCAshunt, identify patients with chronic hepatitis C with cirrhosis,splenomegaly or varices.

In one example, long-term follow-up, may be planned in the HALT C trialin order to determine whether hepatic impairment as defined by QLFTspredicts risk for clinical deterioration.

Example 4. Additional Standard Laboratory Tests

Standard laboratory tests (complete blood count, liver biochemistryprofile) per routine clinical care of the post-hepatectomy donor can beperformed at each center and per the prospective A2ALL Cohort Studydonor protocols. In addition, specific study-related tests can beobtained at times of QLFT testing (baseline, 5 to 10 days, 3 months, and6 months). The latter tests can include: Complete Blood Count; Liverbiochemistry profile (6 month only; others are already included inCohort Study); Body weight; BMI; Medication history (all); and Recordingof any clinical events at 6-month time point.

Example 5. Exemplary Computing Device for Data Analysis

FIG. 1 illustrates an exemplary computing device 100 that can carry outthe operations described herein in accordance with various embodimentsof the present invention. The exemplary computing device 100 isillustrative of many different types of computing devices such as, butnot limited to, a general-purpose computer, a special-purpose computer,web server, and a handheld computer. It is to be understood thatembodiments of the present invention are not limited to the particularcomputing device 100 shown in FIG. 1.

In one embodiment, the computing device 100 is in operable communicationwith a mass spectrometer, which generates chromatographic data. Thechromatographic data can then be transmitted to the computing device100. In another embodiment, the computing device 100 can downloadchromatographic data from a network resource. In yet another embodiment,chromatographic data can be input to the computer via a memory medium,such as a disk. Still another embodiment allows for the chromatographicdata to be manually entered into the computing device 100 (e.g., viakeyboard).

In this simplified example, the computing device 100 comprises a bus orother communication means 101 for communicating information, and aprocessing means such as one or more processors 102 coupled with bus 101for processing information. Computing device 100 further comprises arandom access memory (RAM) or other dynamic storage device 104 (referredto as main memory), coupled to bus 101 for storing information andinstructions to be executed by processor(s) 102. Main memory 104 alsomay be used for storing temporary variables or other intermediateinformation during execution of instructions by processor(s) 102.Computing device 100 also comprises a read only memory (ROM) and/orother static storage device 106 coupled to bus 101 for storing staticinformation and instructions for processor 102. A data storage device107 such as a magnetic disk or optical disc and its corresponding drivemay also be coupled to bus 101 for storing information and instructions.

One or more communication ports 110 may also be coupled to bus 101 forallowing communication and exchange of information to/from with thecomputing device 100 by way of a Local Area Network (LAN), Wide AreaNetwork (WAN), Metropolitan Area Network (MAN), the Internet, or thepublic switched telephone network (PSTN), for example. The communicationports 110 may include various combinations of well-known interfaces,such as one or more modems to provide dial up capability, one or more10/100 Ethernet ports, one or more Gigabit Ethernet ports (fiber and/orcopper), or other well-known interfaces, such as Asynchronous TransferMode (ATM) ports and other interfaces commonly used in existing LAN,WAN, MAN network environments. In any event, in this manner, thecomputing device 100 may be coupled to a number of other networkdevices, clients and/or servers via a conventional networkinfrastructure, such as a company's Intranet and/or the Internet, forexample.

Exemplary Operations for Data Analysis

FIG. 4 illustrates an exemplary model curve derivation algorithm 400having exemplary operations for deriving a model clearance curve inaccordance with a particular embodiment of the present invention. Thealgorithm 400 can be carried out by the computing device 100 shown inFIG. 1. Alternatively, the algorithm 400 could be carried out by adevice other than the computing device 100. Prior to describing thealgorithm 400 in detail, some general aspects of distinguishable agentsand clearance of agents from blood for example are discussed.

With regard to clinical testing with the use of a distinguishable agent,analysis typically involves determining clearance of the agent from abodily fluid or sample such as the blood over time. Clearance generallyrefers to reduction or elimination of an agent concentration in thesample. The clearance can be graphically depicted with an agentconcentration curve, which plots the concentration of the agent withrespect to time. For a given agent, the concentration generally followsa similar curve for different patients. FIGS. 2 and 3 illustratestandard clearance curves that were derived from a sample of over 300patients who were administered cholate orally (FIG. 2) and intravenously(FIG. 3). In this example, fourteen blood samples were taken from eachof the patients to derive the standard clearance curves.

Referring to FIG. 2, the standard oral clearance curve 202 hascharacteristics (e.g., shape) that are generally similar among clearancecurves derived from patients who have ingested cholate. For example, theclearance curve 202 can generally be characterized by a gradual increasein concentration, followed by an exponential decrease. Inflection points204 and 206 are evident in the clearance curve 202. The general shape ofthe clearance curve 202 is characteristic of many agents in addition tocholate. As such, generally clearance curves derived from anyadministered agent such as an oral administration may include inflectionpoints and the general shape as those shown in FIG. 2.

As another example, FIG. 3 is a graph 300 of a clearance curve 302associated with intravenously (IV) administered cholate. The clearancecurve 302 for IV administered cholate is characterized by sudden maximumconcentration 304 around several minutes, followed by exponentialdecline in the concentration. An inflection point 306 generally occurssometime during the exponential decline. The general shape of theclearance curve 302 is typical for most agents that are administeredintravenously. As used herein, an IV clearance curve refers to aclearance curve associated with intravenous administration of an agent,and an oral clearance curve refers to a clearance curve associated withoral administration of an agent.

Although intravenous clearance curves for different agents share thesame general shape and oral clearance curves for different agents sharethe same general shape, they typically differ in some ways. For example,the times at which inflection points occur can differ for differentagents. In addition, the maximum values for agent concentrations candiffer. Also, elimination rates can vary. However, because the clearancecurves have the same general shapes for different agents, useful modelclearance curves can be derived that can be used for conducting tests.Beneficially, such models can reduce the number of blood samples thatneed to be taken from the patients.

With the foregoing in mind, a process can be employed to identifycharacteristics associated with standard clearance curves for adistinguishable agent that is administered to a patient. Thesecharacteristics can be used to derive model clearance curves for futuretests involving the agent. Turning to FIG. 4, the exemplary embodimentof algorithm 400 derives model characteristic curves for IV administeredagent and orally administered agent based on selected times associatedwith characteristics (e.g., inflection points, slope, etc.) of standardIV and oral clearance curves. Although algorithm 400 is described withrespect to cholate, those skilled in the art will recognize that thegeneral process described can be readily adapted to other agents.

In a particular embodiment, prior to executing the algorithm 400, it isassumed that several hundred patients are each administered cholateorally and intravenously. At selected times after the administration ofthe cholate, blood samples are taken from each of the patients. Inaccordance with this embodiment, fourteen blood samples may be takenfrom each of the patients. However, the number of blood samples taken isnot limited to fourteen and may be less or more than fourteen dependingon the application. The fourteen blood samples per patient will be usedto derive a standard fourteen point IV clearance curve and a standardfourteen point oral clearance curve. The blood samples are then preparedto obtain data that is input into the algorithm 400. One samplepreparation protocol is described in Example 2 above.

In one embodiment, step 22 employs gas chromatography mass spectography(GC/MS). For example, a 6890/5973 mass spectrometer from AgilentTechnologies, Inc. can be used. However, other mass spectrometers may beused. In other embodiments, high pressure liquid chromatography-massspectography (HPLC/MS) is employed. For example, an Agilent 1100 seriesLiquid Chromatograph Mass Spectrometer equipped with a G1956A multi-modesource, automatic sampler, HP Chemstation Software or equivalent. TheHPLC-MS can be fitted with a Agilent Eclipse XDB C8, 2.1×100 mm 3.5 umliquid chromatograph column.

The mass spectrometer is instructed (e.g., programmed) to monitor theprepared samples for ions specific to the particular agent of interest.In the embodiment described, the mass spectrometer is programmed tomonitor for ions specific to cholate. In one embodiment, the ionsmonitored are specific to mass fragments of the agent. However, in otherembodiments, other types of ions, such as the molecular ions can bemonitored. The choice of which ions to monitor is dependant upon variousfactors related to the process, including, but not limited to, themolecular size of the agent and how the agent is derivatized.

After the samples are prepared, a receiving operation 402 receives thechromatographic data from the mass spectrometer related to intravenouslyadministered cholate. Another receiving operation 404 receiveschromatograph data related to orally administered cholate. In oneembodiment of the algorithm 400, each of the receiving operations 402and 404 receives fourteen data points representing an average of datafrom fourteen prepared blood samples.

A generating operation 406 generates a standard fourteen point IVclearance curve based on the received IV data. Another generatingoperation 408 generates a standard fourteen point oral clearance curvebased on the received oral data. Those skilled in the art will readilyrecognize how standard fourteen point clearance curves can be generatedin the generating operations 406 and 408.

A deriving operation 410 derives a model IV clearance curve based on thestandard fourteen point IV clearance curve. Another deriving operation412 derives a model oral clearance curve based on the standard fourteenpoint oral clearance curve. Generally, the deriving operations 410 and412 generate model data based on selected data points among the fourteendata points and fit the model data to a curve, referred to as a modelclearance curve. An embodiment of the deriving operation 410 is shown inFIG. 5 and discussed in detail below. An embodiment of the derivingoperation 412 is shown in FIG. 6 and is discussed in detail below.

As discussed, FIG. 5 illustrates a model IV clearance curve derivationalgorithm 500 for deriving a model IV clearance curve based on astandard 14 point IV clearance curve. Referring to FIG. 5, initially thearea under the 14 point IV clearance curve is computed in computingoperation 502. Computing the area under a curve is generally understoodby those skilled in the art. For example, area can be computed usingknown software programs, such as, but not limited to, EXCEL (MICROSOFTCORP.) or MATLAB (THE MATHWORKS, INC.). Alternatively, area can becomputed using a proprietary program. A selecting operation 504 selectssample times corresponding to selected intervals on the standardfourteen point IV clearance curve and/or the standard fourteen pointoral clearance curve. In another embodiment of the selecting operation504, five sample times are selected. In further embodiments, more orfewer than five sample times can be selected. The selecting operation504 can be carried out manually or automatically. In one embodiment,selecting manually involves visually observing the standard 14 pointclearance curve and selecting times within intervals betweencharacteristic points, such as inflection points. To illustrate, theoral clearance curve 202 in FIG. 2 includes four intervals: firstinterval 208, second interval 210, third interval 212, and fourthinterval 214. In FIG. 2, the selected times are indicated by arrowmarkers 216. As shown in this particular embodiment, times at 5 minutes,20 minutes, 45 minutes, 60 minutes, and 90 minutes are selected.

In a computing operation 506, model data is computed that will be usedto generate the model clearance curve. In one embodiment of thecomputing operation 506, agent elimination rates are computed thatcorrespond to each interval shown in FIG. 3. In this embodiment, agentelimination rates are computed using an exponential function. Equation(1) represents an exponential function characteristic of the clearancecurve 302 in FIG. 3:C _(t) =C ₀ e ^(−kt),  Eq. (1)wherein Ct represents the concentration of the agent at time ‘t’, and‘k’ represents the elimination rate.

For each interval, the exponential can be expressed as in Equation (2):C _(ti+1) =C _(ti) e ^(−k(ti−t i+1)),  Eq. (2)wherein ‘i’ represents an interval.

Using Equation (2), the elimination rate for each interval can beexpressed as:k _(i)=ln(Ct _(i+1) /Ct _(i))/(t _(i) −t _(i+1)).  Eq. (3)

In a particular embodiment, four times, 5, 20, 45, and 90, are selectedfor the IV model clearance chart. Each of the selected times correspondsto one of the intervals. Corresponding elimination rates are shownbelow. To determine the first elimination rate, k1, Eq. (3) issimultaneously solved for t₁=5 minutes and t₂=20 minutes resulting inEq. (4):k ₁=ln(C ₂₀ /C ₅)/(t ₁ −t ₂).  Eq. (4)

To determine the second elimination rate, k₂, Eq. (3) is simultaneouslysolved for t₃=45 minutes and t₄=90 minutes resulting in Eq. (5):k ₂=ln(C ₉₀ /C ₄₅)/(t ₃ −t ₁).  Eq. (5)

To determine the function of the model clearance curve between t₂=20minutes and t₃=45 minutes, a third elimination rate, k₃, can be solvedin the same manner as above resulting in Eq. (6):k ₃=ln(C ₄₅ /C ₂₀)/(t ₂ −t ₃).  Eq. (6)

In a fitting operation 508, the elimination rates are used to fit acurve based on five points obtained from the fourteen received IVpoints. In one embodiment, the fitting operation 508 substitutes thecomputed elimination rates, k₁, k₂, and k₃ into Eq. (1) above for eachinterval in order to create a model IV clearance curve.

A computing operation 510 computes the area under the model IV clearancecurve that was fitted in the fitting operation 508. Any of various areacomputation methods may be used as discussed above with respect to thecomputing operation 502.

After the area is computed for the model IV clearance curve, adetermining operation 512 determine whether the area under the model IVclearance curve is within a predetermined range of the area under thestandard 14 point IV clearance curve. In one embodiment, the differencebetween the two areas is computed. The difference in areas is thencompared to a specified threshold. The specified threshold can be set toany value that is applicable to the particular application.

If the determining operation 512 determines that the area under themodel IV clearance curve is not within the predetermined range of thearea under the standard 14 point IV clearance curve, the algorithmbranches ‘NO’ to an adjusting operation 514. The adjusting operation 514adjusts the estimated sample times in a manner to make the two computedareas closer in value. The adjusting operation 514 can be carried outmanually or in an automated fashion.

If the determining operation 512 determines that the area under themodel IV clearance curve is within the predetermined range of the areaunder the standard 14 point IV clearance curve or the algorithm 500 haslooped more than MaxLoops times, the algorithm branches ‘YES’ to anassociating operation 516, which associates the selected times with thedistinguishable agent that was intravenously administered. MaxLoops is aspecified value that is chosen to ensure that looping eventually stopsand sample times are associated with the distinguishable agent.

FIG. 6 is an embodiment of an algorithm 600 for deriving model oralclearance curve based on a standard 14 point oral clearance curve.Initially computing operation 602 computes the area under the standard14 point oral clearance curve. An selecting operation 604 then selectssample times based on the standard 14 point oral clearance curve. Oneembodiment of the selecting operation 604 selects five sample times;however, in other embodiments, the number of sample times may be more orfewer than five sample times.

The estimated five times are generally based on characteristics (e.g.,inflection points) of the standard 14 point oral clearance curve. Theselecting operation 604 can be carried out manually or in an automatedfashion. In some embodiments, the selecting operation 604 can select thetimes derived in the model IV clearance curve derivation 500.

A fitting operation 606 then fits a model clearance curve for the orallyadministered cholate using the five sample times determined in theselecting operation 604. One implementation of the fitting operation 606employs a cubic spline function, as shown in Eq. (7):f _(i)(t)=[f″(t _(i−1))/6(t _(i) −t _(i−1))](t _(i) −t)³+[f″(t _(i))/6(t_(i) −t _(i−1))](t−t _(i−1))³+{[(t _(i−1))/t _(i) −t _(i−1)]−[f″(t ⁻¹)(t_(i−1))/6]}t _(i) −t)+{[f(t _(i))/t _(i) −t _(i−1)]−[f″(t _(i))(t _(i)−t _(i−1))/6]}(t−t _(i−1)),  Eq. (7)wherein f_(i)(t) represents the model clearance curve function duringinterval ‘i’ with respect to time, ‘t’.

Eq. (7) has two unknown second derivatives, f″, for each interval. Tosolve for the two unknown second derivatives, Eq. (7) can bedifferentiated to give an expression for the first derivative for boththe (i−1)th and the ith intervals. Then the two results can be setequal, assuming that the first derivatives at contiguous points on theclearance curve are continuous:f _(i−1)(t _(i))=f _(i)(t _(i)).  Eq. (8)

The following relationship results:(t _(i) −t _(i−1))f″(t _(i−1))+2(t _(i+1) −t _(i−1))f″(t ₁)+(t _(i+1) −t_(i))f″(t _(i+1))=[6/(t _(i+1) −t _(i))][f(t _(i+1))−f(t _(i))]+[6/(t_(i) −t _(i+1))][f(t _(i−1))−f(t _(i))].  Eq. (9)

Also noting the second derivatives at the endpoints are 0, fourequations and four unknowns can be written and solved for all unknownsecond derivatives. After the second derivatives are solved for,complete spline functions could be generated for all 4 intervals. Usingthe above cubic spline equations, the model oral clearance curve isgenerated for intervals 208, 210, 212, and interval 214 up to t=90minutes. (FIG. 2). To generate the portion of the model oral clearancecurve after 90 minutes, an exponential function is used. In oneembodiment, the exponential function after 90 minutes is derived bycomputing the average ‘k’ elimination rate for all sample patients. Theaverage ‘k’ elimination rate is then used in Eq. (1) to generate theremaining portion of the model oral clearance curve. This average ‘k’elimination rate is the same k elimination derived from the IV clearancecurve. In this case, the IV and oral clearance curves decay at the samerate.

After the model oral clearance curve is generated, a computing operation608 computes the area under the model oral clearance curve. Adetermining operation 610 determines whether the area under the modeloral clearance curve is within a predetermined range of the area underthe standard 14 point clearance curve. If the difference between the twoareas is not less than a specified threshold, the algorithm 600 branches‘NO’ to an adjusting operation 612. The adjusting operation 612 adjuststhe sample times to make the two area values closer in value. After thetimes are adjusted, the fitting operation 606 again fits the model datato a model oral clearance curve.

If the determining operation 610 determines that the area under themodel oral clearance curve and the standard 14 point clearance curve arewithin the predetermined threshold or the algorithm 600 has looped morethan MaxLoops times, the algorithm 600 branches ‘YES’ to an associatingoperation 614. The associating operation 614 associates the fiveselected times with the distinguishable agent. FIG. 7 is an exemplarygraph 700 showing a model five point oral clearance curve 702 (dottedline) and a standard 14 point clearance curve 704 (solid line) fromwhich the model curve 702 was derived. Thus, in future tests, thepatient may only need to provide blood samples at less selected timessuch as five selected times (e.g., 5, 20, 45, 60, and 90 minutes), and,using the model oral clearance curve 702, an individualized clearancecurve can be generated for the patient.

To calculate the liver shunt fraction, the exponential decay equationsand the spline function equations, generated mathematically by the 5selected points, are integrated along their respective valid time rangesand an area is generated. The liver shunt fraction is then calculated:ShuntFraction=[AUC_(oral)/AUC_(IV)]*[Dose_(IV)/Dose_(oral)]*100%,  Eq.(10)wherein AUC represents area under the curve and Dose represents theamount (in mg) of dose administered.

The model IV clearance curve derivation algorithm 500 and the model oralclearance curve derivation algorithm 600 may be carried out together.For example, in some embodiments, the algorithms 500 and 600 are carriedout in serial. In other embodiments, the algorithms 500 and 600 arecarried out in parallel. By carrying out the two algorithms together,the sample times for both the IV clearance curve and the oral clearancecurve can be selected so that they are equal. In addition, the order ofoperations described in FIGS. 5-6 are not limited by the orders shown.In some embodiments, operations may be carried out in different orders,and operations may be merged or separated without straying from thescope and spirit of the claimed invention.

Example 6. An Exemplary Clinical and Biochemical Endpoint Study ofDisease Progression

In one example study, two long term studies examined rates of diseaseprogression in patients with HCV with bridging fibrosis and cirrhosis.One study used these estimates to calculate samples sizes for thecurrent NIH treatment trial based upon an equal distribution ofnoncirrhotic and cirrhotic patients. Disease progression can be definedherein as an increase in fibrosis score of 2 points or more, ordevelopment of hepatic decompensation, death from liver disease, or HCC.Table 4 presents a total sample size that would be required to achieve90% power for a binomial chi-square test with a two-sided alpha of 0.05.

TABLE 4 Statistical sample size at various clinical event rates. A B C DControl (%/yr) 4.0 5.0 6.0 7.0 Control (%/4 yr) 15.1 18.5 21.9 25.2 50%decrease in Endpoints IFN (%/yr) 2.0 2.5 3.0 3.5 IFN (%/4 yr) 8.7 10.812.8 14.9 N for 90% power 1084 870 728 626 Noncompliance (5%/yr) IFN(%/4 yr) 9.3 11.5 13.6 15.8 N for 90% power 1324 1064 890 767

During the first 6 months of therapy, all patients can be treated andcontrol and maintenance therapy groups experience disease progression atthe same rate. If the control group has an annual clinical event rate of7% (column D), then 25.2% will have developed a clinical event by theend of four years. If maintenance treatment reduces the annual rate by50% and if treatment is started after 6 months, then the event rate willbe 14.9% at the end of four years. If 5% of the treated group becomenoncompliant each year, then the event rate required to maintainsignificance would be 15.8% at the end of four years. Approximately 1200patients will need to be enrolled into the trial to achieve statisticalsignificance for the primary endpoint. The ability of the study todetermine efficacy for maintenance therapy would be compromised ifeither the rate of development of clinical endpoints is lower thanprojected or if rates of dropout from the trial exceed 5%/yr.

Example 7. Use of Multiple QLFTs in Other Populations (the HALT C Study)

In one example, seven QLFTs were used to define hepatic impairment inpatients with chronic hepatitis C and bridging fibrosis or compensatedcirrhosis enrolled in the Hepatitis Antiviral Long-Term Treatment toPrevent Cirrhosis Trial (HALT C). These results can be compared to thosewith or without biopsy-proven cirrhosis, splenomegaly onultrasonography, and varices at endoscopy.

The mean age of the 248 enrolled patients was 49.9+/−7.3 yr and 75% weremale. Mean BMI was 29.6+/−5.3, 40% had cirrhosis, 60% had bridgingfibrosis, 93% were infected with HCV genotype 1, and mean serum HCV RNAwas 4.39+/−4.66×10⁶ copies/ml. 30% had platelet count<140,000/microliter, 25% had albumin <3.5 g/dl, 25% had INR>1.1, 10% hadbilirubin >1.2 mg/dl, and 25% had AST:ALT>1.

¹³C-methionine (MBT), caffeine (Caf), antipyrine (AP), and2,2,4,4-²H-cholate (CA) were taken orally and 24-¹³C-cholate, galactose(Gal), and lidocaine were administered intravenously. These compounds ortheir metabolites were measured from timed serial samples of blood,saliva, and breath using standard techniques. Elimination rate (kelim),volume of distribution (Vd), clearance (Cl), elimination capacity(Elim), and shunt were calculated from measured analytes. Perfusedhepatic mass (PHM) was determined from SPECT liver scan. Mean testresults were compared by T statistic and area under the receiveroperator curve (ROC) by C statistic. Table 5 presents results ordered byT statistic for association with cirrhosis.

TABLE 5 Results of QLFTs and correlation with various hepaticconditions. % of Pts with Cirrhosis Splenomegaly Varices Abnl T- T- T-Test Test Stat P Stat P Stat P CA Cl_(oral) 70% 7.74 .0000 3.32 .00103.97 .0001 PHM 65% 6.92 .0000 3.93 .0002 4.95 .0000 CA Shunt 75% −6.73.0000 −3.65 .0003 −3.81 .0002 Ca k_(elim) 48% 3.78 .0002 2.33 .0207 1.09NS AP k_(elim) 82% 3.61 .0004 2.56 .0116 2.09 .0399 MBT Score 67% 2.87.0046 3.46 .0007 2.43 .0169 CA k_(elim) 38% 2.86 .0047 1.25 NS 2.36.0195 Gal Elim 73% 2.58 .0106 3.87 .0001 2.28 .0240 AP Cl 58% 2.44 .01601.37 NS 1.84 NS MEGX 15 min 75% 1.33 NS 1.91 .0572 1.88 NS MEGX 30 min67% 1.01 NS 1.77 NS 1.01 NS

PHM had the highest area under ROC with cirrhosis (c-statistic 0.87),splenomegaly (c-statistic 0.75), and varices (c-statistic 0.832) andcorrelated best with platelet count, bilirubin, prothrombin time, andalbumin.

QLFTs may uncover hepatic impairment in fibrotic patients with chronichepatitis C. Certain tests, particularly CA Cloral, PHM, and CAshunt,identify patients with chronic hepatitis C with cirrhosis, splenomegalyor varices. Long-term follow-up may determine whether hepatic impairmentas defined by QLFTs predicts risk for clinical deterioration.

Example 8. Multiple Tests to Assess Hepatic Function

The following example includes multiple tests to assess hepaticfunction. These can include measurement of blood flow with cholateclearance, portal shunt with dual isotope cholate, and microsomalfunction with antipyrine clearance, caffeine clearance, MEGX formationfrom lidocaine and erythromycin breath test. Trough (C1) and peak (C2)concentrations of TAC and MMF concentrations at trough, 1 h, and 2 hpost-dose, relative to dose, can be measured in all recipients.Volumetric studies can be performed using MM, and functional mass willbe measured using the SPECT liver-spleen scan.

Cholate clearance and portal shunt (Blood Flow) involve administrationand measurement of cholate test compounds. Intravenous ¹³C-cholate, forexample 20 mg, can be dissolved in NaHCO₃ solution, passaged through amicropore filter, and placed in sterile, capped glass vials prior touse. This preparation is mixed with 5 ml of 25% human albumin solutionjust prior to intravenous injection. The ²H4-cholate, for example 40 mg,is taken orally. Blood samples for measurement of cholate isotopes canbe obtained at baseline and 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120,150, and 180 minutes post-dose (14 samples, 7 ml red top tubes). Serumconcentrations of cholate are determined by GC/MS-isotope ratiometry.Comparison of intravenous and oral clearance curves allows determinationof first-pass hepatic elimination or portal shunt.

Other tests can be used in combination with the cholate shunt andcholate clearance tests.

Antipyrine and caffeine clearances. Saliva samples, for measurement ofantipyrine, can be obtained at baseline and at 6, 12, 24, 36, 48, and 60hours post-dosing (e.g. 7 samples, mls each). Salivary concentrations ofantipyrine and caffeine are measured by HPLC.

Erythromycin breath test. Breath samples for measurement of ¹⁴CO₂ fromthe metabolism of ¹⁴C-erythromycin are obtained prior to and 20 minutesafter IV administration of ¹⁴C-erythromycin. Breath samples are analyzedfor radioactivity by trapping exhaled CO₂ and liquid scintillationcounting.

MEGX from lidocaine. Blood samples for measurement of MEGX(monoethylglycinexylidide) from the metabolism of lidocaine are obtainedprior to and over 1 hour after the IV administered dose of lidocaine(0.5 mg/kg). MEGX is measured by HPLC.

The data provided by the combination tests will be used to assessoverall organ health and in particular hepatic health. All samples forthe above clearance studies will be coded with a unique identifier,dated, and collection time, center, PI recorded and samples stored intightly-capped vials, and shipped on dry ice to the analyticallaboratory.

Example 9. Determination of Minimal Number of Sample Time Points inCholate Clearance and Cholate Shunt: Development of a “Minimal Model”

Standard methods for measuring oral cholate clearance and shunt,requiring 14 samples of blood collected over 3 h, are clinicallyimpractical. For these reasons mathematical methods were used to definethe minimal sampling requirements.

In this example, a study of patients with chronic hepatitis C was usedand a mathematical model of cholate clearance curves was used togenerate one minimal model necessary to accurately measure cholate shuntin humans. Deconvolutional analysis was utilized on clearance curves ofsimultaneously administered oral and intravenous Doses of 2,2,4,4-²HCholate and 24-¹³C Cholate in order to determine a minimal model forfirst-pass hepatic extraction of cholate in humans.

The following analysis was used to assess the minimal number of timepoints for accurate determination of cholate clearance. Patients (n=286)were enrolled in HALT C trial and participated in the QLFT (quantitativeliver function test) ancillary study; 73 patients were studied twice atdifferent times. Each patient was subjected to the following testprotocol. 20 mg of 24-¹³C cholic acid was dissolved in NaHCO₃, mixedwith 5 ml 25% human albumin solution and injected through an indwellingintravenous catheter over 2 minutes. 40 mg of 2,2,4,4-²H cholic acid wasdissolved in water, mixed in juice and taken orally simultaneously withthe intravenous injection. Blood samples were drawn through theindwelling catheter and taken prior to isotope administration and 5, 10,15, 20, 30, 45, 60, 75, 90, 105, 120, 150 and less than 180 minutespost-dose.

Samples were processed and analyzed by GC/MS as described above toobtain oral and intravenous cholic acid clearance curves. Briefly, serumconcentrations of differentially labeled ¹³C and ²H cholates weredetermined from 0.5 ml aliquots of serum. 1.5 micrograms of unlabelledcholate was added to each serum sample. The cholates were isolated byextraction from serum with Sep-Pak C18 cartridge, acidification, etherextraction, methylation, TMS derivatization, and capillary GC/MS isotoperatiometry, as described above. Cholate shunt was calculated as(AUC_(oral)/AUC_(iv))×(Dose_(iv)/Dose_(oral))×100%. A full descriptionof the methods and mathematical models used in curve fitting,measurement of AUC and analysis of models is provided in thesupplementary Materials section-Appendix 51 of Everson et al., which isincorporated herein by reference in its entirety.

Deconvolutional analysis was performed on the 14-point intravenous andoral clearance curves to obtain the minimal amount of points and timeperiod required to regenerate the full curves. Two to seven time pointsspanning time periods from 5 to 180 minutes were modeled. Meandifferences of measurements (+/−standard deviation) of cholate shuntwith various models using reduced numbers of sample time points incomparison with the 14-point method are shown in FIG. 8. Models usingless than five points were associated with significant mean error andhigher variation. Models incorporating five points or more wereequivalent and within 1-2% of the measurement of cholate shunt by the 14point standard method.

The analysis indicates that cholate shunt may be accurately determinedfrom 5 samples of blood obtained approximately 5, 20, 45, 60 and 90minutes post dose. These time points bracket inflection points in theclearance curves. The accuracy of the 5 point curve, coined a “minimalmodel”, in measurement of cholate shunt was 98.1+1.4% of that calculatedusing all 14 time points. Use of fewer samples or time points that failto bracket inflection points was found to diminish the accuracy ofmeasurement of cholate shunt. The “minimal model” defined by thisanalysis significantly reduces the number of samples and time commitmentrequired to determine cholate shunt in humans. This can improve patientcomfort, compliance with testing, reduce human error in samplecollection and analysis, reduce time and expense, and save resources.The 5-point model greatly simplifies performance of the test increasingits potential for broader application in the assessment of patients withliver disease. Switching from 14 time points to five time points reducespatient phlebotomies by 64% patient sampling time by 50%, and laboratoryanalysis and sample preparation by 64%.

The 5-point model is generally applicable to all test compounds thatexhibit certain characteristics comparable to cholate. Keycharacteristics include relatively high first-pass hepatic elimination,rapid and complete intestinal absorption, retention of the test compoundin the intravascular space, lack of dependency on hepatic metabolism,lack of renal excretion and lack of direct effects of the test compoundon the cardiovascular system or portal circulation. Cholate fulfills allof these criteria. Further validation of cholate as an appropriate testcompound for assessment of portal blood flow and portal-systemic shuntis the fact that the measurement of mean cholate shunt in healthy intacthuman subjects (18%, or 82% first-pass hepatic elimination from theportal vein) is identical to expected, based upon other studies of theliver or liver cells.

Example 10. Liver Metabolic Function: Caffeine Clearance Test

Clearance of caffeine depends upon specific hepatic metabolic pathwaysand its measurement, which quantitates liver metabolic function,requires multiple samples for up to 3 days. One method for measuringdeuterated isotopes of caffeine is described for determining clearancefrom single samples of serum.

Example protocol. In one example, a study was performed to analyzehepatic condition of HCV patients using a multi-isotope method formeasurement of caffeine elimination (TIME test). Caffeine concentrationsrange from 0.1 to 6 micrograms/ml over 24 h after a single oral dose of300 mg. Deuterated caffeine (D3 and D9), unlabeled caffeine, andphenacetin (500 ng/ml) were added to five separate samples of calf serumand extracted after alkalinization using methylene chloride. Themethylene chloride layer was taken to dryness and reconstituted in 50microlites of acetone. Compounds were analyzed by GC-MS with an initialoven temperature of 40° C. for 0.55 min, increasing at 50° C./min to280° C., held isothermally at 280° C. for 4 min, and quantified byselected ion monitoring (m/z 179, 194, 197, and 203) using calibrationcurves with phenacetin as internal standard.

Example outcome. The correlation coefficients for the calibration curveswere 0.995, 0.996 and 0.995 for unlabelled, D3 and D9 caffeines,respectively.

X+/−SD and coefficients of variance (CV) for unlabeled caffeine (2800ng/mL) and D3 & D9 (400 ng/mL each) were: 2800+/−109, 3.9%; 411+/−18,4.4%; and 385+/−16 ng/ml, 4.2%, respectively. Instrument precision was99.50%, 99.38%, and 99.51%, respectively. These concentrations reflectexpected concentrations in human serum 4 h after an oral dose of 300 mgof total caffeine at a molar ratio D3 (or D9):unlabeled caffeine of 1:7.

X+/−SD, and CV of unlabeled caffeine (600 ng/mL) and D3 & D9 (150 ng/mLeach), were 539+/−61, 11%; 143+/−12, 8.4%; and 135+/−16 ng/mL, 12% withprecision of 98.73%, 99.43% and 99.22%, respectively. Theseconcentrations reflect expected concentrations in serum 24 h after anoral dose of 300 mg total caffeine with a molar ratio of D3 (orD9):unlabeled caffeine of 1:4. This example method accurately quantifiescaffeine and deuterated isotopes over concentration ranges achievedafter oral dosing with 300 mg caffeine.

A triple isotope method (TIME test) by performance of appropriateclinical testing of human subjects and comparison of the results tostandard caffeine clearance assays can be evaluated as follows:

TABLE 6 TIME assay study groups. Validation Study Subjects: Group 1:Healthy controls (N = 10) Group 2: HCV patients, Ishak fibrosis stage0-2 (N = 10) Group 3: HCV patients, Ishak fibrosis stage 4-6 (N = 10)

Protocol A: Subjects are placed on a caffeine-free diet for 3 days thenadmitted to a monitoring center such as GCRC. Baseline samples of blood,serum and saliva for measurement of for example caffeine, CBC, INR,Chemistry profile (creatinine, liver tests included), pregnancy test anda history and physical examination.

TABLE 7 Administration of caffeine and caffeine isotopes. UnlabelledIsotope 1 Time = t1 Isotope 2 Time = t2 Isotope 3 Time = t3

Post-dose samples were obtained as Sample 1, 2, 3, 4 and 5. Repeat thestudy (items 1-5 above) after washout, 24 h< washout <7 d.

Protocol B: Same as Protocol A, but with no caffeine-free diet. Subjectsare admitted to a monitoring center such as GCRC. Baseline samples ofblood, serum and saliva for measurement of for example caffeine, CBC,INR, Chemistry profile (creatinine, liver tests included), pregnancytest and a history and physical examination.

Methods:

1. Addition of phenacetin as internal standard.

2. Extraction of caffeine and caffeine isotopes from samples.

3. Standard caffeine analysis by HPLC.

4. Caffeine isotopes are measured by GC/MS or HPLC/MS.

Calculations:

1. Multiple sampling: Ln/linear regression of [caffeine] vs time.Slope=elimination rate constant. Intercept yields [caffeine] at t=0, Volof distribution calculated. Clearance product of elimination rate andvol of distribution.

2. Single samples (TIME test). Each sample is analyzed for concentrationof each of the 3 isotopes. Sample time is difference between time ofisotope administration and time of collection. Ln/linear regression of[caffeine] vs time, yields elimination rate, vol dist, and Cl.

Statistics

1. Compare elim rate, vol dist, and Cl between standard and TIMEmethods, using Protocol A data. 2. Compare effect of dietary caffeine onboth standard and TIME methods by comparing results for each methodbetween Protocol A and Protocol B. 3. Define reproducibility of standardand TIME methods by comparing the initial and repeat studies done inboth protocol A and protocol B.

The TIME test may be used alone or in combination with a cholate shuntassay or one or more other QLFTs to provide a comprehensive assessmentof hepatic condition. Similarly, this test could be used to assessimpact of disease, disease progression, therapies, interventions ortransplantation.

Example 11. Impact of Virological Response on Hepatic Function

The goal of this study was to determine the relationships ofquantitative liver function tests (QLFTs) with virological responses topeginterferon (PEG)+/−ribavirin (RBV) in patients with chronic hepatitisC. Serial QLFTs were used to define the spectrum of hepatic improvementafter sustained virological response (SVR).

Rates of sustained virological response (SVR) withpeginterferon/ribavirin treatment of chronic hepatitis C are lower inpatients with advanced hepatic fibrosis or cirrhosis. In the Hepatitis CAntiviral Long-term Treatment against Cirrhosis (HALT-C) Trial, patientswith chronic hepatitis C with bridging fibrosis or compensated cirrhosis[Child-Turcotte-Pugh (CTP)≤6] and prior nonresponse were retreated withpeginterferon/ribavirin. In this cohort, SVR after retreatment declinedstepwise, from 23% to 9%, with increasing severity of disease, asdefined by liver histology and platelet count. Because quantitativeliver function tests (QLFTs) measure the continuum of liver impairment,it was reasoned that the relationship between SVR and disease severitymight be better defined by QLFTs. Sustained virological response reduceshepatic inflammation, fibrosis, and rates of clinical outcomes. Theprincipal clinical manifestations of advanced chronic hepatitis C, suchas varices, ascites and encephalopathy are linked to portal hypertensionand impaired hepatic function. Beneficial effects of SVR on hepaticfibrosis and clinical outcomes are probably mediated throughimprovements in the portal circulation and hepatic function—improvementswhich could be detected by QLFTs, but not by standard laboratory tests.

In this study of retreatment of patients with chronic hepatitis C withpeginterferon/ribavirin, a battery of QLFTs was utilized to measurehepatic metabolism, hepatic and portal blood flow, portal-systemicshunting and hepatic parenchymal mass. One goal was to define therelationships between severity of hepatic impairment, as measured byQLFTs and virological responses. In addition, serial QLFTs were used todefine hepatic improvement after achievement of SVR.

Participants (n=232) were enrolled in the Hepatitis C AntiviralLong-term Treatment against Cirrhosis (HALT-C) Trial, had failed priortherapy, had bridging fibrosis or cirrhosis and were retreated withPEG/RBV. All 232 patients had baseline QLFTs; 24 patients with SVR and68 nonresponders had serial QLFTs. Lidocaine, [24-¹³C]cholate, galactoseand ^(99m)Tc-sulfur colloid were administered intravenously;[2,2,4,4-²H]cholate [1-¹³C]methionine, caffeine and antipyrine wereadministered orally. Clearances (Cl), breath ¹³CO₂,monoethylglycylxylidide monoethylglycylxylidide (MEGX), perfused hepaticmass (PHM) and liver volume were measured as described above and inEverson et al., 2009 “Quantitative tests of liver function measurehepatic improvement after sustained virological response: results fromthe HALT-C trial”, Aliment. Pharmacol. Ther. 29, 589-601, which isincorporated herein by reference.

Results

Rates of SVR were 18-26% in patients with good function by QLFTs, butless than or equal to 6% in patients with poor function. Results areshown in FIG. 10 which shows the percentage change between baseline andfollow-up studies for various QLFTs. The black bars depict the changesafter SVR, and the grey bars show the changes in patients withnonresponse (NR). Compared to patients with nonresponse, patientsexperiencing SVR had significant improvements in caffeine and antipyrineelimination rates (k_(elim)), antipyrine clearance (Cl), clearance oforally administered cholate (Cl_(oral)), cholate shunt and perfusedhepatic mass (PHM). Hepatic metabolism, as measured by caffeine k_(elim)(P=0.02), antipyrine k_(elim) (P=0.05) and antipyrine clearance (P=0.02)improved after SVR. The portal circulation, as measured by cholate oralclearance (P=0.0002), cholate shunt (P=0.0003) and PHM (P=0.03), alsoimproved significantly after SVR.

Cholate oral clearance and cholate shunt, both measurements of portalcirculation, improved significantly after SVR. Cholate oral clearanceand cholate shunt at baseline and post-SVR are shown in FIG. 9.Sustained virological response was associated with a 32% increase incholate oral clearance, a measure or portal blood flow, as shown in FIG.9(a). Sustained virological response was also associated with a 26%decrease in cholate shunt, a measure of portal-systemic shunting, asshown in FIG. 9(b).

Example 12. Cholate Shunt, IV and Oral Cholate Clearance Tests withAnalysis by HPLC-MS

Collection and Processing of Samples.

Reagents and Supplies.

The following reagents and supplies are utilized in the Cholate Shuntand Cholate Clearance Test procedures. If the patient is undergoing onlythe oral cholate clearance test, the IV Solution and 25% Human Albuminfor injection are omitted.

IV Solution-20 mg 24-¹³C-Cholic Acid in 5 cc 1 mEq/ml Sodium Bicarbonate

PO test compounds 2,2,4,4-²H (40 mg) and Sodium Bicarbonate (600 mg)

25% Human Albumin for injection (5 mls) to be added to 24-¹³C-CholicAcid solution.

IV supplies, including 250 mls NS, indwelling catheter, 3-way stopcock.

10 cc syringes for administering IV test compounds

7 cc red top tubes for sample collection

3 ml crovials for serum storage

Needle discard bucket

Apple or Grape (non-citrus) juice for oral test compounds

Timer

Centrifuge

Transfer Pipets

Patient Preparation.

It is ascertained that the patient has no allergic reaction to latex. Itis further ascertained that the patient has had nothing to eat or drink(NPO), except water, since midnight the night before the test day. Thepatient height and weight are measured and recorded. The patient isfitted with an IV with a three-way stopcock and normal saline to keepopen (NS TKO) is placed before the test begins.

Cholate Compound Stock Solutions.

Test Compound Preparation.

The Oral Solution is utilized for either or both of the oral cholateclearance test and/or the cholate shunt assay. The oral solutionconsisting of 2,2,4,4-²H-Cholic acid (40 mg) and Sodium Bicarbonate (600mg) is dissolved in about 10 cc water 24 hours prior to testing bymixing vigorously. The solution is stored in either the refrigerator orat room temperature. Just prior to administration, grape or apple(non-citrus) juice is added to the mixture. The juice solution is mixedwell and poured into cup for patient to drink. The cup is rinsed withextra juice which is administered to the patient.

The IV Solution is utilized for either or both of the IV cholateclearance test and/or the cholate shunt assay. A formulation of 20 mgCholic Acid-24-¹³C in 5 cc 1 mEq/ml Sodium Bicarbonate is prepared bypharmacy staff. The Test dose is 20 mg Cholic Acid-24-¹³C in 10 ccdiluent. If vial is frozen, it is allowed to thaw completely. Just priorto beginning the test, the Cholic Acid-24-¹³C solution is mixed withalbumin as follows (this method prevents loss of test compound duringmixing process). Draw up all of 24-¹³C-Cholic Acid solution (about 5 cc)in a 10 cc syringe. Draw up 5 cc albumin in another 10 cc syringe.Detach needle from the 24-¹³C-cholate syringe and attach a 3-waystopcock. Detach needle from albumin syringe and inject albumin throughstopcock into 24-¹³C Cholate syringe. Draw a little air into the bileacid/albumin syringe and mix solutions gently by inverting syringeseveral times. Expel air.

Test Compound Administration.

Collect baseline samples before test compounds are given. The time thesespecimens are collected should be recorded on sample collection recordsheet. Administration of test compounds is performed as follows. Starttimer. Record 24 hour clock time as T=0. Record time. At T=1-3 minutesadminister oral compounds. Have the patient drink the oral solution andjuice. Rinse cup with more juice and have patient drink rinse. Recordtimer time. At T=4-5 minutes-using the 3-way stopcock administer the IVpush of 20 mgs ¹³C Cholic acid in 5 mls 25% Human Albumin. Record timertime. Return line to NS through 3-way stopcock.

Specimen Collection.

Collect all samples via the 3-way stopcock with 0.5 ml discard beforeeach sample to prevent dilution or cross-contamination of samples.Collect 5 ml red tops at the following times. (T=timer time).

-   -   a. T=10 minutes, collect 5 minute, record timer time;    -   b. T=25 minutes, collect 20 minute, record timer time;    -   c. T=50 minutes, collect 45 minute, record timer time;    -   d. T=65 minutes, collect 60 minute, record timer time;    -   e. T=95 minutes, collect 90 minute, record timer time.        Specimen Handling.

Red top tubes are allowed to clot at room temperature for at least 30minutes. All blood tubes are spun for 10 minutes at 3000 rpm. Serum isremoved to properly labeled vials and frozen at −20° C. until samplesare transported.

Preparation of Cholate Compound Stock Solutions.

Accurate determination of cholate clearances and shunt is dependent onaccurate calibration standards. Concentrations of cholic acid compoundsin stock solutions must be accurate and reproducible. Very accurate(error <0.5%) portions of the cholic acid powders are weighed and glassweighing funnels and washes of 1 M NaHCO₃ are used to ensurequantitative transfer of the powder to the flask. Volumetric flasks areused to ensure accurate volumes so that the final concentrations of theprimary stock solutions are accurate. Calibrated air displacementpipettes are used to dispense accurate volumes of the primary stocksolutions that are brought to full volume in volumetric flasks toprepare secondary stock solutions that are also very accurate. Secondarystock solutions are used to prepare the standard curve samples, accuracyand precision samples, recovery samples, quality control samples,selectivity samples, and stability samples as described in theappropriate SOPs.

The following reagents are required.

1 M NaHCO₃

0.1 M NaHCO₃

0.1 M NaHCO₃/2% BSA

Methanol, LCMS grade

Water, CLRW grade (Clinical Laboratory Reagent Water)

Cholic Acid, purity 98%

Chenodeoxycholic Acid, purity 98%

[24-¹³C]-Cholic Acid, 99 atom % ¹³C

[2,2,4,4-²H]-Cholic Acid, 98 atom % ²H.

All primary stock solutions are prepared at a concentration of 250 uMusing Table 8 below.

TABLE 8 Cholate compound primary stock solutions. 13-C Chenodeoxcholiccholic acid cholic acid 4-D cholic acid acid MW 408.56 409.59 412.60392.56 purity 98.0% 99.0% 98.0% 98.0% volume 100 ml 100 ml 100 ml 100 mlconc 250 uM 250 uM 250 uM 250 uM weight 10.42 mg 10.34 mg 10.53 mg 10.01mg

Primary stock solutions are prepared separately in 0.1 M NaHCO₃ and inmethanol as follows. Weigh out the appropriate amount of cholic acidcompound (+/−0.05 mg) in a glass weighing funnel. Transfer the powder toa 100 ml volumetric flask. Use either methanol or 0.1M NaHCO₃ to rinseany residual powder from the funnel into the flask. Bring to a finalvolume of 100 ml with methanol and mix well. Label flask with anexpiration of 1 month. Store at −20° C.

The unlabeled cholic acid is prepared as a 50 uM internal standard ineither MeOH or 0.1 M NaHCO₃ as follows. Pipette 2.0 ml of theappropriate 250 uM CA primary standard into a 10 ml volumetric flask.Bring to a total volume of 10 ml with 0.1 M NaHCO₃ or methanol and mixwell. Label flask with an expiration of 1 year. Store at 4° C.

[24-¹³C]-Cholic Acid secondary stock solutions made in methanol areshown in Table 9. Each secondary stock solution into the appropriate 15ml glass screw top test tube. Tubes are securely capped and sealed withseveral layers of parafilm and stored at −20° C.

TABLE 9 [24-¹³C]-Cholic acid secondary stock solutions in methanol.Final assay Secondary 250 uM Concentration Stocks 13C-CA (m) MethanolTotal uM uM ul ml ml 0.20 B (m) 2.0  80 +  9.92 = 10.00 1.00 D (m) 10.0 400 +  9.60 = 10.00 6.00 F (m) 60.0 2400 +  7.60 = 10.00 2880 27.1230.00

[2,2,4,4-²H]-Cholic Acid secondary stock solutions made in methanol areshown in Table 10. Each secondary stock solution into the appropriate 15ml glass screw top test tube. Tubes are securely capped and sealed withseveral layers of parafilm and stored at −20° C.

TABLE 10 [2,2,4,4-²H]-Cholic acid secondary stock solutions in methanol.Final assay Secondary 250 uM Concentration Stocks 4D-CA (m) MethanolTotal uM uM ul ml ml 0.30 I (m) 3.0  120 +  9.88 = 10.00 1.00 K (m) 10.0 400 +  9.60 = 10.00 3.00 L (m) 30.0 1200 +  8.80 = 10.00 1720 28.2830.00

[24-¹³C]-Cholic Acid secondary stock solutions made in 0.1 M NaHCO₃ andBSA are shown in Table 11. Each secondary stock solution is transferredinto the appropriate 15 ml screw top plastic tube, capped, sealed withseveral layers of parafilm and stored at 4° C.

TABLE 11 [24-¹³C]-Cholic acid secondary stock solutions in 0.1M NaHCO₃and BSA. Final assay Secondary 250 uM 0.1M 2% Concentration Stocks13C-CA NaHCO3 BSA Total uM uM ul ml ml ml 0.10 A 1.0  40 + 4.96 + 5.00 =10.00 0.20 B 2.0  80 + 4.92 + 5.00 = 10.00 0.60 C 6.0  240 + 4.76 + 5.00= 10.00 1.00 D 10.0  400 + 4.60 + 5.00 = 10.00 2.00 E 20.0  800 + 4.20 +5.00 = 10.00 6.00 F 60.0 2400 + 2.60 + 5.00 = 10.00 10.00 G 100.0 4000 +1.00 + 5.00 = 10.00 7960   27.04   35.00   70.00

[2,2,4,4-²H]-Cholic Acid secondary stock solutions made in 0.1 M NaHCO₃and BSA are shown in Table 12. Each secondary stock solution istransferred into the appropriate 15 ml screw top plastic tube, capped,sealed with several layers of parafilm and stored at 4° C.

TABLE 12 [2,2,4,4-²H]-Cholic acid secondary stock solutions in 0.1MNaHCO₃ and BSA. Final assay Secondary 250 uM 0.1M 2% ConcentrationStocks 4D-CA NaHCO3 BSA Total uM uM ul ml ml ml 0.10 H 1.0  40 + 4.96 +5.00 = 10.00 0.30 I 3.0  120 + 4.88 + 5.00 = 10.00 0.50 J 5.0  200 +4.80 + 5.00 = 10.00 1.00 K 10.0  400 + 4.60 + 5.00 = 10.00 3.00 L 30.01200 + 3.80 + 5.00 = 10.00 5.00 M 50.0 2000 + 3.00 + 5.00 = 10.00 3960  26.04   30.00   60.00

The secondary stock solutions as prepared above are utilized inpreparation of accuracy and precision samples in human serum withunlabeled cholate as an internal standard. The secondary stock solutionsare used in preparation of recovery samples with addition of unlabeledcholate as an internal standard.

In order to accurately measure patient liver function with the cholateshunt assay, the two different stable isotope cholate compounds musteach be accurately quantified in patient serum. In order to do this, theaccuracy, precision, and recovery of each of the two standard curvesmust be validated over their respective ranges of concentrations.

The accuracy and precision of an assay are assessed by running multiplereplica samples at the lower limit of quantification (LLOQ), low,medium, and high range of concentrations. Accuracy is the closeness ofthe average measured value to the actual value. Precision is thereproducibility of the measured value as indicated by the CV. Therecovery is assessed by comparing the detector response of the analyteextracted from serum relative to that of pure analyte measured at low,medium, and high concentrations.

Preparation of Quality Control Samples

The FDA provides guidance as to acceptable levels of accuracy andprecision of analytical methods. See, for example, Bioanalytical MethodValidation, May 2001, Section VI. Application of Validated Method toRoutine Drug Analysis. Once the analytical method has been validated forroutine use, its accuracy and precision should be monitored regularly toensure that the method continues to perform satisfactorily. To achievethis objective, a number of QC samples are prepared separately andshould be analyzed with processed test samples at intervals based on thetotal number of samples. The QC samples are run in duplicate at threeconcentrations (one near the lower limit of quantification (LLOQ) (i.e.,3×LLOQ), one in midrange, and one close to the high end of the range)and should be incorporated in each assay run. The number of QC samples(in multiples of three) will depend on the total number of samples inthe run. The results of the QC samples provide the basis of accepting orrejecting the run. At least four of every six QC samples should bewithin 15% of their respective nominal value. Two of the six QC samplesmay be outside the 15% of their respective nominal value, but not bothat the same concentration.

The QC samples must cover the high, middle, and low ranges of bothstandard curves. The QC samples are designed to closely simulate theactual concentrations of labeled compounds found in patient serum overthe time course of the testing. The [24-¹³C]-CA concentration is veryhigh at the early time point and falls exponentially to medium and lowconcentrations. The [2,2,4,4-²H]-CA concentration is very low at theearly time point, rises to its highest value in the middle time pointsand then falls to a medium concentration.

Supplies

The following supplies are utilized to prepare the QC samples used inthe Cholate Shunt and Cholate Clearance Test procedures. If the patientsamples are undergoing only the oral cholate clearance test, the[24-¹³C]-CA QC samples can be omitted.

Human Serum AB (Gemini Bio-Products #100-512) Unlabeled Cholate InternalStandard Stock Solution (IS; 50 uM Cholic Acid in 0.1M NaHCO₃)

[24-¹³C]-Cholic Acid and [2,2,4,4-²H]-Cholic Acid Secondary StockSolutions in 0.1 M NaHCO₃/1% BSA:

B  2.0 uM [24-¹³C]-CA D 10.0 uM [24-¹³C]-CA F 60.0 uM [24-¹³C]-CA I  3.0uM [2,2,4,4-²H]-CA K 10.0 uM [2,2,4,4-²H]-CA L 30.0 uM [2,2,4,4-²H]-CA10 ml volumetric flasksP1000 air displacement pipette and 1 ml tipsNew, clean cryovialsProcedure for Preparation of Quality Control Samples for CholateClearance and Assays.

The [24-¹³C]-Cholic Acid and [2,2,4,4-²H]-Cholic acid QC samples areprepared as follows. For each set of QC samples, label 3 clean 10 mlvolumetric flasks as “QC 1”, “QC 2”, and “QC 3” as shown in Table 13.Larger volumetric flasks can be used to prepare larger batches. Use 1/10the nominal volume of the larger flasks as the amount of secondary stocksolution to add as indicated below.

TABLE 13 QC samples. Tubes [24-¹³C]-CA [2,2,4,4-²H]-CA QC1 1.00 ml F1.00 ml I QC2 1.00 ml D 1.00 ml L QC3 1.00 ml B 1.00 ml K

Using a P1000, add 1.0 ml of the appropriate [24-¹³C]-CA stock solutionand 1.0 ml of the appropriate [2,2,4,4-²H]-CA stock solution to theappropriate flasks as indicated in Table 13. Bring each flask to anexact total of 10.0 ml with human serum. Securely cap each flask and mixwell by inversion several times. Label 8 cryovials as “QC 1”, 8 as “QC2”, and 8 as “QC 3”. Aliquot 1.2 ml of each QC mixture into theappropriate vials. Store the QC samples frozen at −80° C. QC sampleshave an expiration of 1 year.

High Pressure Liquid Chromatography-Mass Spectroscopy (HPLC-MS) SamplePreparation

In order to ensure accurate liver function testing, the labeled cholatetest compounds must be isolated and identified from patients' serumsamples. Cholate compounds are amphipathic molecules with bothhydrophobic and hydrophilic regions. Cholates are also carboxylic acidsthat can exist in either an uncharged free acid form (cholic acid) or acharged carboxylic acid form (cholate) depending on pH. These propertiescan be exploited to isolate cholate compounds from serum. The use ofHPLC/MS as opposed to GC/MS, allows analysis of cholate without samplederivitization.

Reagents, Supplies and Equipment

The following reagents are prepared and used in the HPLC-MS samplepreparation.

Water, CLRW grade (Clinical Laboratory Reagent Water)

Methanol, LCMS grade

Diethyl Ether, ACS grade

Unlabeled Cholic Acid Internal Standard (IS) Primary Stock Solution (50uM CA in 0.1 M NaHCO₃)

Quality Control Samples (prepared as described above)

1.0 N NaOH (dissolve 20 g NaOH in 500 ml water)

0.01 N NaOH (dilute 1.0 N NaOH 1 to 100 with water)

10% Methanol (add 100 ml Methanol to a 1 L cylinder and bring to 1.0 Lwith water)

90% Methanol (add 900 ml Methanol to a 1 L cylinder and bring to 1.0 Lwith water)

0.2 N HCl (add 1.0 ml ACS grade Concentrated HCl slowly with stirring to57.0 ml water)

Mobile Phase (10 mM Ammonium Acetate/60% Methanol)

Disposable 16×100 and 13×100 test tubes

P1000 air displacement pipette and 1 ml tips

P100 air displacement pipette and 0.2 ml tips

Repeater Pipette

Vortex Mixer

SPE cartridges (Bond Elut LRC C18 OH, 500 mg, Varian, Inc)

Vacuum Manifold

Speed-Vac

Benchtop centrifuge

Speed-Vac vented to fume hood

Bath Sonicator

Repeater Dispensers for water, methanol, 10% methanol, and 90% methanol

Remove patient serum samples and a set of QC samples (2 each of QC1, 2,and 3) from the freezer and allow them to thaw to room temperature.Personal protective equipment (PPE) including lab coat, gloves, eyeprotection must be worn. All eluates and equipment must be disinfected.Pipettes and tips that come in contact with the sample must be discardedinto hazardous waste.

Label a set of test tubes (16×100) for each patient with that patient'sinitials and the time point code (5 min is 1, 20 min is 2, 45 min is 3,60 min is 4, 90 min is 5). Using a P1000 pipette, transfer 0.50 ml ofpatient's serum from the appropriate collection tube into theappropriate test tube.

Label a set of test tubes (16×100) for each QC sample (QC1a, QC1b, QC2a,QC2b, QC3a, QC3b). Using a P1000, transfer 0.50 ml of each QC sampleinto the appropriate test tube.

Label 2 test tubes (13×100) as STD1 and STD2.

To each patient sample and each QC sample and each STD sample tube, add50 ul of the Unlabeled Cholic Acid Internal Standard (IS) Primary StockSolution using a Repeater Pipette.

Set aside the STD tubes for later acidification and ether extraction instep 21.

To each patient sample tube and QC sample tube add 1.0 ml of 0.01 N NaOHwith a Repeater pipet and vortex 30 sec.

Label a set of SPE cartridges with one for each patient serum and QCsample to be processed.

In the hood add 5 ml Methanol with a repeater dispenser to eachcartridge. This step may be done on a vacuum manifold with high vacuumor by gravity. This wets the resin bed with solvent. Once the top of theliquid reaches the top of the frit add the next solution. Avoid lettingthe cartridges run dry.

Add 10 ml Water with the repeater dispenser to each cartridge. Thisequilibrates the resin bed to prepare it for binding cholate compounds.This step may be done on the vacuum manifold on high vacuum or bygravity.

To each SPE cartridge add the appropriate sample. The cholate compoundswill bind to the resin bed. To each sample test tube add a 1 ml waterrinse with the repeater, vortex, and add this rinse to the appropriatecartridge. Allow the sample to run by gravity for 20 minutes or longerthen may use low vacuum ≤3 inches Hg to pull sample through.

After the sample has completely entered the resin bed, add 2.5 ml Waterto each SPE cartridge with the repeater dispenser. This washes thecolumn resin bed. Use low vacuum ≤3 inches Hg.

To each SPE cartridge add 2.5 ml 10% Methanol with the repeaterdispenser. This further washes the column resin bed. Use low vacuum ≤3inches Hg.

Label a set of test tubes (13×100) with one for each patient sample andeach QC sample.

Place each test tube in a rack and on top place its matching SPEcartridge.

To each SPE cartridge add 2.5 ml 90% Methanol with the repeaterdispenser. This elutes the cholate compounds which are collected intothe test tubes.

Place the test tubes in the Speed-Vac and centrifuge under vacuum withhigh heat for 45 min to reduce eluate volume and to remove methanolwhich interferes with ether extraction.

To each tube from the Speed-Vac and to each of the STD tubes, add 0.5 mlof 0.2 N HCl with the Eppendorf Repeater Pipette and vortex 30 sec. Thisacidification converts the cholate compounds into their free acid formfor ether extraction.

In the fume hood, to each tube add 3 ml of diethyl ether and vortexvigorously for 30 sec. This extracts the free acid form of the cholatecompounds into the ether phase.

Centrifuge 5 minutes at a minimum of 5000 rpm to accelerate phaseseparation.

Label another set of test tubes (13×100) one for each sample.

Carefully collect the upper ether layer and transfer to the new testtubes.

Place the ether extracts in the Speed-Vac vented to the fume hood andcentrifuge under vacuum without heat until samples are dry.Alternatively, samples can be dried with a gentle stream of N₂ gas.

Add 100 ul Mobile Phase to dried samples, vortex 30 sec and sonicate.

Transfer samples to Agilent 1.5 ml vials and cap.

HPLC/MS Parameters and System Preparation

Reagents, Supplies and Equipment

The following reagents are prepared and used in the HPLC-MS sampleanalysis.

Water, Clinical Laboratory Reagent Water (CLRW)

Methanol LCMS grade

10 mM Ammonium Acetate water

10 mM Ammonium Acetate methanol

Mobile Phase: 60% 10 mM Ammonium Acetate Methanol/40% 10 mM AmmoniumAcetate

Water

Volumetric flasks, appropriate sizes

Graduated cylinder

The following instruments and supplies are used in the HPLC-MS sampleanalysis.

Calibrated analytical balance

HPLC/MS instrument: Agilent 1100 series Liquid Chromatograph MassSpectrometer equipped with a G1956A multi-mode source, automaticsampler, HP Chemstation Software or equivalent.

Agilent Eclipse XDB C8, 2.1×100 mm 3.5 um liquid chromatograph column

Solvent Filter Degasser

0.22 μm nylon filters

The solvents and mobile phase are each prepared, filtered with a 0.22 μmnylon filter and degassed. Solvents and mobile phase each expire 48hours after preparation.

The LCMS system is prepared and tuned; the column is stabilized at 40°C. and conditioned by running the mobile phase for 30 min. The samplesare loaded to the autosampler. The column flow rate is 0.4 ml/min ofisocratic mobile phase buffer; 60% 10 mM Ammonium Acetate Methanol/40%10 mM Ammonium Acetate Water. 5 microliters of each sample is injectedby the autosampler. The MS is run in multimode electrospray (MM-ES)ionization with atmospheric pressure chemical ionization (APCI)ionization. Selected ion monitoring is performed at 407.30, 408.30 and411.30 m/z. Peaks are integrated by the system software. Three QCsamples are assayed with each analytical run. The concentration of theQC samples must fall within 15% accuracy.

Example 13. Hepatic Impairment Measured by Quantitative Tests of LiverFunction (QLFTs) Predicts Clinical Outcome in Patients with AdvancedFibrosis

Liver biopsy is the current standard for defining disease severity andpredicting risk for clinical outcomes in patients with chronic hepatitisC. But, biopsy is invasive, inconvenient, risky, and prone to samplingerror. QLFTs noninvasively measure hepatic impairment and correlate withbiochemical and histological indices of disease severity. The ability ofQLFTs to predict clinical outcomes in compensated patients was evaluatedprospectively, controlling for platelet count and histologic cirrhosis.

In this example, 227 patients, enrolled in the HALT-C Trial, hadbaseline QLFTs and were randomized to either no treatment (N=120) orpeginterferon alfa-2a (PegIFN) 90 μg/week (N=107), and followed for 66months for non-HCC, liver-related clinical outcomes. Since PegIFN didnot affect clinical or histological outcomes (DiBisceglie et al., Dec.4, 2008, Prolonged therapy of advanced chronic hepatitis C with low-dosepeginterferon, NEJM, 359:2429-2441), these two groups were combined foranalyses. QLFTs included cholate clearance po, methionine breath test,cholate shunt, antipyrine clearance, perfused hepatic mass, liver andspleen volume from SPECT liver-spleen scan, cholate clearance iv,caffeine elimination rate, MEGX concentration, and galactose eliminationcapacity (GEC). The hazard ratio (HR) of clinical outcomes was based oncomparing the third of patients with worst function to those with mediumfunction and medium function to high function. Results are shown inTable 14. The independence of each QLFT in predicting clinical outcomeswas assessed in multivariate analyses that included platelet count andhistologic cirrhosis.

Results of the trial showed 46 of the 227 patients (20%) experienced 97outcomes: CTP score ≥7 (N=34), death (N=30), ascites (N=18), varicealbleed (N=3), encephalopathy (N=2), and SBP (N=1).

TABLE 14 Hazard Ratio (HR) of Clinical Outcome for Various QLFTs. HR pertertile (95% CI) QLFT Univariate Multivariate Cholate Clearance po 4.37(2.63-7.30) 3.23 (1.84-5.65) Methionine Breath Test 3.06 (1.83-5.13)2.42 (1.43-4.12) Cholate Shunt 3.26 (2.06-5.17) 2.35 (1.44-3.84)Antipyrine Clearance 2.59 (1.57-4.29) 2.30 (1.39-3.80) Perfused HepaticMass 3.62 (2.26-5.81) 2.12 (1.26-3.89) Spleen Volume 3.45 (2.15-5.53)1.92 (1.12-3.31) Cholate Clearance iv 2.13 (1.43-3.17) 1.83 (1.23-2.73)Caffeine Elimation 2.24 (1.48-3.38) 1.81 (1.20-2.73) MEGX 1.89(1.29-2.78) 1.67 (1.14-2.45) Galactose Elimination Capacity 1.89(1.29-2.78) 1.67 (1.14-2.45) HR for clinical outcomes between lowest andhighest tertile is HR squared.

Cholate Clearance po, Methionine Breath Test, Cholate Shunt, AntipyrineClearance, Perfused Hepatic Mass, and Spleen Volume were the most robustindependent predictors of clinical outcomes. The cutoff values for oralcholate clearance (HepQuant-Oral) and cholate shunt (HepQuant-Dual) fordefining the group of HCV patients at greatest risk for future hepaticdecompensation were: Oral Cholate Clearance <11 mL/(min kg); and CholateShunt ≥39%. It was concluded that measurement of hepatic impairment byQLFTs outperforms histologic cirrhosis in prediction of clinicaloutcomes and provides a noninvasive means for assessing disease severityand counseling patients.

Example 14. Interval Clinical Outcomes in Relation to the Results ofQLFTs at Each Visit

In this example, Hepatitis C patients from the HALT-C trial wereassessed by oral cholate clearance and cholate shunt assays at 0, 24 and48 months. Patients were monitored over a six year period. The cholateshunt (CA Sh) and oral cholate clearance (CA Cl oral) were determined atthe beginning of each time interval. Cutoffs for cholate shunt thatpredicts the 2-year risk of decompensation were determined for cholateshunt and oral cholate clearance assays. “No outcome” or “none” in FIGS.11-14 refers to no outcome prior to or within the follow-up (f/u)interval. “Outcome” refers to a first clinical outcome that occurredwithin the follow-up interval.

FIG. 11 shows average cholate shunt values and the number of hepatitis Cpatients at each time interval who experienced first clinical outcome,or no outcome, in the periods 0 to 24 months, 24 to 48 months and after48 months. The cutoff for cholate shunt that predicts the 2-year risk ofdecompensation is shown in FIG. 12.

FIG. 13 shows average oral cholate clearance values and the number ofhepatitis C patients at each time interval who experienced firstclinical outcome, or no outcome, in the periods 0 to 24 months, 24 to 48months and after 48 months. The cutoff for oral cholate clearance thatpredicts the 2-year risk of decompensation is shown in FIG. 14.

Example 15. Cholate Shunt is Predictive of Ishak Fibrosis Score

Hepatitis C patients (n=282) from the HALT-C trial were assessed bycholate shunt assay and liver biopsy. The patients were fairly evenlydistributed across the fibrosis stages. Results are shown in FIG. 15. AsIshak fibrosis score increases, cholate shunt increases. The error barsindicate 95% CI limits for the means of Ishak fibrosis score. Mean (+/−)for cholate shunt for fibrosis stages 2-6 were 27+/−9%; 33+/−13%;37+/−15%; 44+/−; and 51+/−17%, respectively. Table 15 shows valuesderived from the equation fitting the mean values in FIG. 15; 95% CI wasdefined from the standard errors for cholate shunt at each Ishak stage.The average 95% CI based on standard errors was +/−10% of the mean.

TABLE 15 Use of Cholate Shunt Test Results as an Estimate of Stage(Ishak) of Hepatic Fibrosis. Ishak Fibrosis Score Shunt Result 95% CI20% 0   0 to 0.6 25% 1.4 0.7 to 2.0 30% 2.6 1.9 to 3.2 35% 3.6 2.9 to4.2 40% 4.4 3.8 to 5.1 45% 5.2 4.5 to 5.8 50% 5.9 5.2 to 6   >50% 6

At a cholate shunt of 30%, the mean Ishak fibrosis score was 2.6. Thecholate shunt value was found to be predictive of the Ishak fibrosisscore by about +/−0.5 fibrosis units; roughly the variation seen whentwo pathologists read the same biopsy. Cholate shunt can potentially beused as a non-invasive alternative to liver biopsy in determination ofIshak fibrosis score. See Everson et al., “The spectrum of hepaticfunctional impairment in compensated chronic hepatitis C: results fromthe Hepatitis C Anti-viral Long-term Treatment against Cirrhosis Trial”,Aliment. Pharmacol. Ther. 27, 798-809.

Example 16. A Kit for Determination of Cholate Shunt and IV and OralCholate Clearance

A kit containing the following components can be provided to thephysician for the purpose of administering differentially labeled stablecholate isotopes for determination of oral cholate clearance, IV cholateclearance and cholate shunt. The kit also provides certain supplies forpatient sample collection. Test compound preparation, patientadministration and sample collection are performed according to packageinsert directions. Patient samples are prepared according to packageinsert directions. See for example, the testing protocol in Example 12,above. The samples are frozen and shipped to a central referencelaboratory for analysis. An analytical report is generated by thereference laboratory and can be sent to the physician by e-mail, fax ormail. In this example, 11 components are provided in a single kit asoutlined in Table 16.

TABLE 16 Kit contents. For IV Administration 1 ¹³C Cholic Acid inbicarbonate solution [USP] 1 Bottle (20 mg in 5 ml, sterile) 2 HumanSerum Albumin [USP] (5 ml 25% w/v, sterile) 1 Bottle For OralAdministration 3 Powdered ²H Cholic acid (40 mg powder or in 1 Bottlesolution) For Blood Sample Collection, Serum Separation, & Transport 4Labeled Blood-Serum Collection Tubes (6 count, 6 Tubes sterile) 5Labeled Transport Vials with Internal Cholic Acid 6 Tubes Standard (6count, 0.5 ml minimum volume) Containers and Shipping 6 Shipping/Samplereturn box (Use same box) 1 Box 7 Box label 1 Label 8 Return mailinginstructions insert 1 Insert 9 Package insert 1 Insert 10 Return label 1Label 11 Prepaid return shipping 1 Label

In another example, the kit comprises an oral dose of a distinguishablecholate and sample tubes for collection of samples over a period of lessthan 3 hours after administration of the distinguishable agents. Inanother example, a kit may comprise components necessary for a testperiod of 90 minutes post administration of an isotopically labeledcholic acid.

All of the COMPOSITIONS and/or METHODS and/or APPARATUS disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variation may beapplied to the COMPOSITIONS and/or METHODS and/or APPARATUS and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

I claim:
 1. A method for assessment of hepatic function in a subjecthaving, or suspected of having or developing, a hepatic disorder, themethod comprising: (a) receiving a plurality of blood or serum samplescollected from the subject following oral administration of a dose of afirst distinguishable compound (dose_(oral)) to the subject andintravenous co-administration of a dose of a second distinguishablecompound (dose_(iv)) to the subject, wherein the samples have beencollected over intervals of no more than seven time points spanning atime period consisting of no more than 180 minutes after administration;(b) quantifying the concentration of the first and the seconddistinguishable compounds in each sample by a method comprisinghigh-pressure liquid chromatography-mass spectrometry (HPLC-MS); (c)generating an individualized oral clearance curve from the concentrationof the first distinguishable compound in each sample comprising using acomputer algorithm curve fitting to a model oral distinguishablecompound clearance curve and computing the area under the individualizedoral clearance curve (AUCoral); (d) generating an individualizedintravenous clearance curve from the concentration of the seconddistinguishable compound in each sample by use of a computer algorithmcurve fitting to a model intravenous distinguishable compound clearancecurve and computing the area under the individualized intravenousclearance curve (AUCiv); and (e) calculating the liver shunt fraction inthe subject using the formula;AUCoral/AUCiv×Doseiv/Doseoral×100%; and (f) comparing the liver shuntfraction in the subject to a distinguishable compound liver shuntfraction cutoff value, wherein the liver shunt fraction in the subjectcompared to the cutoff value is an indicator of the hepatic function ofthe subject.
 2. The method of claim 1, wherein the first and seconddistinguishable compounds are compounds that are extracted from theportal blood in its first pass through the liver by at least about 60%,80%, or 85% in healthy controls.
 3. The method of claim 1, wherein thefirst and second distinguishable compounds are distinguishable bileacids or xenobiotics.
 4. The method of claim 3, Wherein the first andsecond distinguishable bile acids are distinguishable cholate compoundsindependently selected from the group consisting of distinguishablecholic acids, distinguishable glycine-conjugated cholic acids,distinguishable taurine-conjugated cholic acids, distinguishablechenodeoxycholic acids, distinguishable glycine-conjugatedchenodeoxycholic acids, and distinguishable taurine-conjugatedchenodeoxycholic acids.
 5. The method according to claim 3, wherein thefirst and second distinguishable bile acids are stable isotope labeleddistinguishable cholate compounds.
 6. The method of claim 1, wherein thesamples were collected from the subject over a period of about 90minutes or less.
 7. The method of claim 6, wherein the samples werecollected from the subject at about 5, 20, 45, 60, and 90 minutespost-dose.
 8. The method of claim 1, wherein the comparing of the livershunt fraction in the subject to the cutoff value is used to assess aneed for at least one therapeutic treatment of the subject with ahepatic disorder when the shunt fraction is above the cutoff value. 9.The method of claim 8, wherein the at least one therapeutic treatment isselected from the group consisting of antiviral therapy, interferontherapy, chemotherapeutic agents, endoscopic therapy, transjugularintrahepatic portosystemic shunt (TIPS) placement, hepatic resection,and liver transplantation.
 10. The method of claim 8, wherein thehepatic disorder is selected from the group consisting of chronichepatitis C, chronic hepatitis B, hepatocellular carcinoma (HCC),primary sclerosing cholangitis (PSC), cirrhosis, splenomegaly, fibrosisstage, varices, liver transplant rejection, delayed function of livertransplant, recurrent disease in transplanted graft, liver injury, andprolonged infection.
 11. The method of claim 1, further comprising atleast one additional hepatic assessment test.
 12. The method of claim11, wherein the at least one additional hepatic assessment test isselected from the group consisting of clearance or metabolism ofaminopyrine, clearance or metabolism of antipyrine, clearance ormetabolism of bile acids other than cholate, clearance or metabolism ofcaffeine, clearance of or metabolism erythromycin, clearance ormetabolism of nitroglycerin, clearance of or metabolism galactose,clearance or metabolism of indocyanine green, clearance or metabolism oflidocaine, clearance or metabolism of midazolam, clearance or metabolismof omeprazole, clearance or metabolism of dextromethorphan, clearance ormetabolism of phenacetin, clearance or metabolism of methacetin,clearance or metabolism of methionine, ultrasonography, elastography,magnetic resonance imaging (MRI) elastography, liver-spleen scan, serumbilirubin analysis, alanine aminotransferase analysis, aspartateaminotransferase analysis, alkaline phosphatase analysis, prothrombinanalysis, creatinine analysis, platelet count, blood count analysis,serum albumin and MELD (model for end-stage liver disease) score. 13.The method of claim 1, wherein the step of comparing the liver shuntfraction in the subject to the shunt cutoff is used to assess anincreased risk for future clinical outcome of at least one hepaticdisorder in the subject when the shunt fraction is above the cutoffvalue.
 14. The method of claim 13, wherein the cutoff value for shuntfor defining a hepatitis C patient at greatest risk for future hepaticdecompensation is liver shunt fraction ≥39%.
 15. The method of claim 1,wherein the shunt cutoff value is derived from normal healthy controls,within a given individual over time, patients who respond to therapy,patients with large varices, patients with sustained virologicalresponse to antiviral therapy, patients unable to respond to antiviraltherapy, patients with significant fibrosis, or patients with cirrhosis.